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Bioaccumulation: terrestrial

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Endpoint:
bioaccumulation: terrestrial
Type of information:
calculation (if not (Q)SAR)
Remarks:
Migrated phrase: estimated by calculation
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: no analytical dose confirmation,
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Earthworm were cultivated to investigate the toxic effect of different nanomaterials on reproduction according to OECD 222. After that the earthworms were depurated for 24 h on filter paper and freazed for the analysis of the titanium concentration in the whole earthworm after 56 days of exposition.
GLP compliance:
yes
Radiolabelling:
no
Details on sampling:
Ti was determined in the earthworms. Earthworms were incubated for 24 h on wet filter paper
to purge their guts. Afterwards they were frozen (-20°C) until analysis.
Vehicle:
no
Test organisms (species):
Eisenia fetida
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: Regenwurmfarm Tacke, Borken, Germany
- Age at test initiation (mean and range, SD): 2-12 month
- Weight at test initiation (mean and range, SD): 250-600 mg (wm)

ACCLIMATION
- Acclimation period: 7d
- Acclimation conditions (same as test or not): not the same soil (artificial soil)
Total exposure / uptake duration:
28 d
Total depuration duration:
24 h
Test temperature:
20 ± 2 °C
pH:
test start: 4.8 - 5.4
test end: 6.2 - 6.9
TOC:
no data
Moisture:
no data
Details on test conditions:
TEST SYSTEM
- Test container (material, size): polypropylene containers (Bellaplast GmbH, Alf)
- Amount of soil or substrate:640 g soil (dw) + 40 g cow dung (ww)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE
- Sampling depth (cm):5 cm
- Soil texture (if natural soil)
- % sand: 71
- % silt: 24
- % clay: 5
- Soil ID: RefeSol 01-A, batch IME-01
- Organic carbon (%):Org C: 0.93
- Maximum water holding capacity (in % dry weigth): 55% of the maximum water-holding capacity
- Pretreatment of soil: The soil was sieved to ≤ 2 mm. The soil was not sterilised.
- Storage (condition, duration): The soil had been stored outdoors in high-grade stainless steel basins with drainage and ground contact at the test facility at 4°C.

OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle of 16 h:8 h
- Light intensity: 600 lux

TEST CONCENTRATIONS
- Test concentrations: additional concentrations in 2nd and 3rd test
2nd and 3rd test with P25: 50, 100, 200, 500, 750, 1000 mg/kg soil, dry mass (application via powder on soil)
2nd test with NM 101 and NM 103: 50, 100, 200, 400 mg/kg soil, dry mass (application via powder on soil).
Nominal and measured concentrations:
Nominal concentrations:
P25:
50, 100, 200, 500, 750, 1000 mg/kg soil, dry mass (loading: soil)
10, 20, 50, 100, 200 mg/kg soil, dry mass (loading: food and soil)

NM 101 and NM 103:
10, 20, 50, 100, 200, 400 mg/kg soil, dry mass (loading: food and soil)
Type:
BSAF
Value:
0.1 other: kg soil / kg earthworm (depurated)
Basis:
whole body d.w.
Remarks:
; P25
Remarks on result:
other: Geometric mean (Min: 0.02kg/kg, Max: 0.4 kg/kg)
Type:
BSAF
Value:
0.15 other: kg soil/ kg earthworm (depurated)
Basis:
whole body d.w.
Remarks:
; NM-101
Remarks on result:
other: Geometric mean (Min: 0.11 kg/kg; Max: 0.23 kg/kg)
Type:
BSAF
Value:
0.08 other: kg soil/kg earthworm (depurated)
Basis:
whole body d.w.
Remarks:
; NM-103
Remarks on result:
other: Geometric mean (Min: 0.02 kg/kg; Max: 0.38 kg/kg)
Kinetic parameters:
Kinetics were not examined.

Table 1: Estimated titanium concentration in earthworm after exposition to soil with different titanium dioxide nanomaterials spiked in soil or food and the calculated biota-sediment accumulation factor (BSAF)

Test TiO2nanomaterial

Application route

Sample n-Ti (mg /kg)

Mean (mg/kg)

SD (mg/kg)

BSAF
(organism/ environment)

P25 (1. test)

-

control

58.4

9.8

0.17

P25 (1. test)

soil

6

54.2

4.7

0.09

P25 (1. test)

soil

12

77.4

2.9

0.04

P25 (1. test)

soil

30

76.4

19.9

0.26

P25 (1. test)

soil

60

76.3

12.7

0.17

P25 (1. test)

soil

120

72.4

29.0

0.40

P25 (1. test)

food

6

58.6

4.3

0.07

P25 (1. test)

food

12

64.6

4.1

0.06

P25 (1. test)

food

30

75.3

6.1

0.08

P25 (1. test)

food

60

101

7.0

0.07

P25 (1. test)

food

120

121

31

0.26

P25 (2. test)

-

control

55.2

2.2

0.04

P25 (2. test)

soil

30

49.2

4.3

0.09

P25 (2. test)

soil

60

43.7

1.5

0.03

P25 (2. test)

soil

120

50.1

11.9

0.24

P25 (2. test)

soil

300

61.0

1.4

0.02

P25 (2. test)

soil

600

88.0

21.7

0.25

NM-101 (1. test)

-

control

54.1

-

-

NM-101 (1. test)

soil

6

28.9

4.9

0.17

NM-101 (1. test)

soil

12

38.1

4.1

0.11

NM-101 (1. test)

soil

30

70.8

7.5

0.11

NM-101 (1. test)

soil

60

53.1

12.3

0.23

NM-101 (1. test)

soil

120

52.7

8.6

0.16

NM-101 (1. test)

food

6.0

49.7

5.0

0.10

NM-101 (1. test)

food

12

45.1

3.7

0.08

NM-101 (1. test)

food

30

59.4

10.4

0.18

NM-101 (1. test)

food

60

66.2

1.7

0.03

NM-101 (1. test)

food

120

107

40

0.37

NM-103 (1. test)

soil

control

22.6

2.2

0.10

NM-103 (1. test)

soil

6

55.3

21.1

0.38

NM-103 (1. test)

soil

12

30.1

1.5

0.05

NM-103 (1. test)

soil

30

30.8

1.5

0.05

NM-103 (1. test)

soil

60

32.9

1.1

0.03

NM-103 (1. test)

soil

120

31.1

2.3

0.07

NM-103 (1. test)

food

6

33.8

6.3

0.19

NM-103 (1. test)

food

12

43.6

9.6

0.22

NM-103 (1. test)

food

30

23.6

3.0

0.13

NM-103 (1. test)

food

60

56.1

3.4

0.06

NM-103 (1. test)

food

120

62.9

1.0

0.02

Table 2: Calculated BSAF values based on the mean titanium concnetration in earthworm and the concentration in the tested substrate spiked with titanium via food or soil.

TiO2 test nanomaterial Application
 route
BSAF Min
(kg soil/kg earthworm)
BSAF Max
(kg soil/kg earthworm)
BSAF Geom. Mean (kg soil/kg earthworm)
P25 soil 0.02 0.40 0.10
P25 food 0.06 0.26 0.10
NM-101 soil 0.11 0.23 0.15
NM-101 food 0.03 0.37 0.11
NM-103 soil 0.03 0.38 0.08
 NM-103  food  0.02  0.22  0.09
Validity criteria fulfilled:
not specified
Conclusions:
Earthworms (Eisenia fetida) were exposed to 10 - 1000 mg/kg of different TiO2-NP (rutile/anatase, rutile, anatase) in artificial substrate for 28 days. After a depuration period of 24 h, whole body burdens were determined. For the different nanomaterials, median biota-soil accumulation factors range from 0.08 -0.15 kg soil/kg earthworm and median biota-food accumulation factors range from 0.09 -0.11 kg food/kg earthworm indicating that earthworms do not bioaccumulate TiO2-NP via soil or food. Titanium in the earthworms was not localised and an adsorption of TiO2-NPs to the earthworms, however, cannot be excluded.
Endpoint:
bioaccumulation: terrestrial
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: no measurement of exposure concentrations, data are displayed in graphs only
Reason / purpose:
reference to same study
Qualifier:
no guideline required
Principles of method if other than guideline:
Wheat was grown in a sandy-soil mixtures spiked with TiO2-NP for 84 d. After the harvest, Ti concentrations of roots and grains were measured.
GLP compliance:
not specified
Radiolabelling:
no
Details on sampling:
Sampling for grain analysis:
- Sampling intervals/frequency for test organisms: Harvest after 84 d of growth.
- Plants were dried at 70 °C, grains were extracted and ground in a ball mill (MM 400, Retsch, Haan, Germany) at a frequency of 25 s(-1) for 4 min.
- Soil samples were dried at 105 °C.

Sampling for root analysis:
- Prior fixation/embedding process, samples were rinsed three times with PBS.
- Fresh root samples were pre-fixed in 2.5% Glutaraldehyde in phosphate buffered saline (PBS) after harvesting and stored at 4°C.
- After incubation with 1% Osmium tetroxide at room temperature (RT) for 40 min samples were rinsed with water three times before they were incubated with 1% Uranyl acetate dihydrate in water for 1 h and rinsed with water three times again.
- Dehydration was performed with 50% (15 min), 70% (20 min), 90% (25 min), 100% (5 min) and 100% water free ethanol for 30 min and Propylene oxide 100% for 30 min.
Vehicle:
no
Details on preparation and application of test substrate:
Prepared in two 300 g sand-soil mixtures:
- 300 g of a 50% (v/v) sand-soil mixture was weighed into a 500 mL Schott bottle and 0.03 g, 3 g or 30 g of test substance were added
- Zinc sulphate was grinded to powder in a mortar prior to addition in order to allow for homogenous mixing
- Sand, soil and test substance were then mixed in a powder mixer (Turbula® T 2 F, Willy A. Bachofen AG, Basel, Switzerland) for 30 min.

Treatment of pre-mixture in cement mixer.:
- pre-mixture were added to 30 kg of a sand-soil mixture (50 % v/v) to final test substance concentrations of 1, 100 or 1000 mg n-TiO2/kg
- the mixing chamber run for 6 hours in a slightly raised position
- soil was not dried and concentrations, therfore the sand-soil mixture was slightly moist resulting in somewhat higher concentrations of 1044, 104 and 1.04 mg/kg dry weight (dw), respectively.




Test organisms (species):
other: Triticum ssp.
Details on test organisms:
TEST ORGANISM
- Common name: Wheat
- Age at test initiation: seeds
Total exposure / uptake duration:
84 d
Test temperature:
25 °C (light)/ 16°C (dark)
pH:
7.7
TOC:
no data
Moisture:
na data
Details on test conditions:
TEST SYSTEM
- Test container: shortened PVC-sewer pipe
- Amount of soil or substrate: 4 kg mixed soil
- No. of organisms per container (treatment): 6 at test start, 3 after emergence (day 5)
- No. of replicates per treatment group: 2
- No. of replicates per control: 2
- Watering: plants were watered 3 times/wk by refilling the soil content to 60 % WHC

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site (latitude, longitude): N47° 25' 39.564"; E8° 31' 20.04"
- Treatments with fertilizers: 7.9 mL Hoagland solution (10 kg/ha) applicated once in the first four weeks, once a week from week five
- Depth of sampling: 1 cm
- Other: Backround concentration of Ti: 783 mg/kg
- Soil description: brown earth with sandy loamy to loamy fine fraction
- pH water: 7.7
- Humus content (%): 0.55
- Cation exchange capacity (CEC): 6.0 cmol+/kg
- Maximum water holding capacity (in % dry weigth): 31

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark
Nominal and measured concentrations:
nominal concentrations: 1, 100, and 1000 mg n-TiO2/kg soil
measured concentration: 1.04, 104, and 1004 mg n-TiO2/kg soil
Reported statistics:
Statistical analysis was done with Rstudio (R Core Team, 2013). Data was tested for homoscedasticity and normality using the Barlett’s test and Shapiro–Wilk test, respectively. In case of homoscedastic and normally distributed data, analysis of variance was conducted with a one-way ANOVA and Dunnet’s test was performed for comparison of treatments with the control. If data was not homoscedastic and/or normally distributed, Mann-Whitney test was performed for comparison. In any case, level of significance was 5%.
If the blocks showed significant differences between each other, generalized linear model of gaussian family was fitted to include the error of the block.
Data was nominated and tested for correlation among variables. If correlations with Pearson's r greater than |0.8| were found, one variable was removed before performing a principal component analysis. Removed were the variables N, residual ears, residual flag leafs, shoot to root ratio and number of grains. A principal component analysis was conducted with the remaining data.

TiO2-NPs could not be found in wheat roots in any of the treatments.There were indications for TiO2 NPs and aggregates in thin sections of roots from the bulk TiO2 treatment but it remains inconclusive if particles were taken up by the root or if it is an experimental artefact.

Ti concentrations in grains were significantly lower at the 1 mg/kg TiO2 -NP treatments compared to the control. At treatments with 100 and 1000 mg/kg TiO2 -NP, Ti concentrations of grains were variable and differences are not statisticallly different. Thus, it remains unclear whether wheat accumulates Ti in the grain at elevated TiO2 concentrations in soil.

Validity criteria fulfilled:
not specified
Conclusions:
TiO2 NPs were not found in wheat roots. Regarding the translocation of Ti to grains in wheat at elevated TiO2 concentrations in soil, results are not conclusive.
Endpoint:
bioaccumulation: terrestrial
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline but well documented study.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Ti concentrations in plant leaves were measured after 54 d of growth
GLP compliance:
not specified
Test organisms (species):
other: Rape (Brassica napus), Cabbage (Brassica oleraccea) and Red Fescue (Festuca rubra).
Details on test organisms:
Seeds of rape were bought from Gondian SA (54 district, France).
Seeds of cabbage were produced by RAGT (54 district, France).
Seeds of red fescue were produced by Carneaux freres SA (54 district, France).
They were stored at 4°C.
Total exposure / uptake duration:
54 d
Test temperature:
16-24°C
pH:
7.1±0.2
Details on test conditions:
Rape, cabbage and red fescue were grown on sediment in which mercury was previously labelled with its radioactive isotope 203Hg.
Pots were arranged in a growth chamber with light intensity of 500 µmolphotons/m²s and 16h photoperiod. Pots were watered daily and sediment solutions weekly collected after 12h of stabilisation using soil moisture samplers.
Leaves were harvested after 54 days of growth, dried at 25°C and finely ground in agate vessels.
Type:
BSAF
Value:
0.001 dimensionless
Basis:
other: leaves
Calculation basis:
other: after 54 d
Remarks on result:
other: Brassica napus; Concentration environment: 3040 mg Ti/kg dw, concentration leaves: 2.4 mg Ti/kg dw.
Type:
BSAF
Value:
0.001 dimensionless
Basis:
other: leaves
Calculation basis:
other: after 54 d
Remarks on result:
other: Brassica oleraccea; Concentration environment: 3040 mg Ti/kg dw, concentration leaves: 2.0 mg Ti/kg dw
Type:
BSAF
Value:
0 dimensionless
Basis:
other: leaves
Calculation basis:
other: after 54 d
Remarks on result:
other: Festuca rubra; Concentration environment: 3040 mg Ti/kg dw, concentration leaves: 0.7 mg Ti/kg dw
Kinetic parameters:
No information on kinetics

Titanium concentration of rape (B. napus), cabbage (B. oleraccea) and red fescue (F. rubra) leaves:

B. napus: 2.4 mg/kg dw

B. oleraccea 2.0 mg/kg dw

F. rubra: 0.7 mg/kg dw

Validity criteria fulfilled:
not specified
Conclusions:
BSAF values for titanium range from 0.0002 to 0.0008 kg/kg for leaves of 3 different plant species (rape, cabbage and fescue) grown for 54 d on dredged sediment in the laboratory containing 3040 +/- 210 mg Ti/kg dw.

Description of key information

 Data available for uptake of micro- and nanosized TiO2 by different plant and earthworm species indicate an absence of a bioaccumulation and biomagnification potential.

Key value for chemical safety assessment

Additional information

Microsized TiO2:

BSAF values of titanium range from 0.0002 to 0.0008 kg/kg for leaves of 3 different plant species (rape, cabbage and fescue) grown for 54 days on dredged sediment in the laboratory containing 3040 +/- 210 mg Ti/kg dw. Hence, Ti has a low potential for bioaccumulation in plants (Caille et al. 2005).

This low uptake of Ti in plants is assumed to be due to the low solubility of microsized TiO2 at environmentally relevant conditions.

Nanosized TiO2:

According to the recent ECHA guidance on aquatic bioaccumulation (Appendix R 7-2 Recommendations for NM applicable to Chapter R7c) it is not possible to predict the bioaccumulation of nanomaterials from the log Kow or solubility. Hence, BCF/BAF values have to be experimentally determined. Further, for nanomaterials that undergo dissolution it is recommended to obtain information on the form of the substance as present in the animal tissue. Since nano-TiO2 does not to dissolve under environmentally relevant conditions, a potential uptake is unlikely to be associated with the uptake of dissolved Ti forms. Thus, nanosized TiO2 (if any) could potentially only be taken up of via adsorption to biological surfaces, direct penetration, or endocytic processes such as via phagocytosis, pinocytosis, and caveolae-dependent or clathrin-mediated endocytosis.

In several laboratory bioaccumulation studies with TiO2-NP and plants and earthworms exposed to spiked soil, accumulation kinetics were not addressed and it was not always clarified if steady-state conditions were reached.

In a supporting study by Wyrwoll et al. (2014), earthworms (Eisenia fetida) were exposed to 1000 mg/kg of different n-TiO2 materials in a sandy soil for 28 days followed by a depuration period of 24 h. Whole body burdens of earthworms exposed to TiO2 were below control burdens and indicate that earthworms do not bioaccumulate TiO2-NP. Similar conclusions can be drawn from reliable studies of the same species exposed to different concentrations of TiO2-NP in artificial substrate and spiked food (Hund-Rinke & Klawonn, 2013).

Further supporting bioaccumulation studies indicate a low terrestrial bioaccumulation potential of nanosized TiO2:

Lapied et al. (2011) exposed Lumbricus terrestris for 2-8 weeks to a TiO2 nanocomposite (TiO2 core coated with superposed layers of Al(OH)3 and PDMS) mixed into food or soil at concentrations ranging from 0 to 100 mg/kg. Based on Ti localization by XRF microscopy, nanoparticles appear to be rather inert and do not cross the intestinal epithelium/ chloragogenous matrix barrier to enter the coelomic liquid, or the cuticle barrier to reach the muscular layers.

Bioaccumulation of tomato plants (fruit, root, stem and leaves) after a 5-weeks exposure to 1000 and 5000 mg/kg TiO2-NPs in potting soil was low, resulting in BAFs ranging from 0.00062 to 0.0946 (Song et al. 2013).

Servin et al. (2013) applied qualitative μ-XRF and μ-XANES analysis to examine root-to-fruit translocation of TiO2-NP in cucumber suggesting that TiO2-NP could be introduced into the food chain with unknown consequences. However, controls were not examined and concentrations were not quantified so that a contamination cannot be excluded.

In sum, data available for the uptake of TiO2-NP or titanium by different plant and earthworm species indicate a low bioaccumulation and biomagnification potential.