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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: no quantification of titanium concentration of test soils
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
GLP compliance:
no
Remarks:
, GLP principles followed, no archiving of the raw data, qualitiy assurance unit not involved. All laboratory equipment was controlled and documented according to GLP.
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
SPIKING OF SOIL WITH TIO2 POWDER
- Direct addition of the test substance into test vessel: yes; the TiO2 material was mixed with 1% of the total amount of air-dried test soil. This part was subsequently mixed with a spoon to the rest of the test oil
- Solvents: no

SPIKING OF FEED WITH TIO2 POWDER
- Direct addition of the test substance into test vessel: yes; air-dried, ground cow dung was mixed with TiO2 powder. Thereafter, the dung was moistend with deionized water.
- Solvents: no

SPIKING OF SOIL WITH AQUEOUS TIO2 DISPERSION
- Direct addition of the test substance into the test vessel: no
- Sonication of dispersion: yes; The TiO2 material was added to deionized water (100 and 200 mg/L), stirred with a magnetic flea (900 rpm; 1 min) and sonicated for three minutes in a bath sonicator. Subsequently, 250 ml of the dispersion was sprayed onto the soil and mixed thoroughly with the test soil.
- Solvents: no
- Appearance of dispersion: no information
- Test medium: deionized water

SPIKING OF FEED WITH AQUEOUS TIO2 DISPERSION
- Direct addition of the test substance into the test vessel: no
- Sonication of dispersion: yes; The TiO2 material was added to deionized water (212 or 424 mg/L), stirred with a magnetic flea (900 rpm; 1 min) and sonicated for three minutes in a bath sonicator. Subsequently, 120 ml of the dispersion was applied on the dry dung and mixed thoroughly with the dung, resulting in TiO2 soil concentrations of 10 and 20 mg/kg dw soil.
- Solvents: no
- Appearance of dispersion: no information
- Test medium: deionized water
Test organisms (species):
Eisenia andrei
Animal group:
annelids
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), organisms were fed with cow manure
Study type:
laboratory study
Substrate type:
natural soil
Total exposure duration:
56 d
Post exposure observation period:
There was no post exposure period.
Test temperature:
20 ± 2 °C
pH:
test start: 4.8 - 5.4
test end: 6.2 - 6.9
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): Loamy, medium acidic, and lightly humic sand (RefeSol 01A)
- % sand: 71
- % silt: 24
- % clay: 5
- Soil ID: RefeSol 01-A, batch IME-01
- Organic carbon (%):Org C: 0.93
- Maximum water holding capacity: 227 ml H2O/kg
- 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
- Once per week, the water content was checked gravimetrically and evaporated water was replaced. Every 7 days, 20 g (wet weight, corresponding to 5 g dry weight) of uncontaminated food was spread on the soil surface in each container.

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:
Only nominal concentrations:

P25:
50, 100, 200, 500, 750, 1000 mg/kg soil, dry mass (application via powder on soil)
50, 100, 200 mg/kg soil, dry mass (application via powder on feed)
10, 20 mg/kg soil, dry mass (application via dispersion on soil)
10, 20 mg/kg soil, dry mass (application via dispersion on feed)
First test:
NM 101 and NM 103:
50, 100, 200, 400 mg/kg soil, dry mass (application via powder on soil)
50, 100, 200 mg/kg soil, dry mass (application via powder on feed)
10, 20 mg/kg soil, dry mass (application via dispersion on soil)
10, 20 mg/kg soil, dry mass (application via dispersion on feed)
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25, particle size: 21 nm
Basis for effect:
mortality
Remarks on result:
other: powder-spiked soil
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25, particle size: 21 nm
Basis for effect:
other: biomass
Remarks on result:
other: powder-spiked soil
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25, particle size: 21 nm
Basis for effect:
reproduction
Remarks on result:
other: powder-spiked soil
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
>= 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 101, particle size: 8 nm
Basis for effect:
mortality
Remarks on result:
other: powder-spiked soil
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
>= 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 101, particle size: 8 nm
Basis for effect:
other: biomass
Remarks on result:
other: powder-spiked soil
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
>= 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 101, particle size: 8 nm
Basis for effect:
reproduction
Remarks on result:
other: powder-spiked soil
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
>= 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 103, particle size: 20 nm
Basis for effect:
mortality
Remarks on result:
other: powder-spiked soil
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
>= 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 103, particle size: 20 nm
Basis for effect:
reproduction
Remarks on result:
other: powder-spiked soil
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
>= 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 103, particle size: 20 nm
Basis for effect:
other: biomass
Remarks on result:
other: powder-spiked soil
Details on results:
Reported NOEC values refer to tests in which no stimulation of reproduction of E. andrei was observed.

P25, NM 101 and NM 103: NOEC values (survival, growth & reproduction)

≥ 200 mg/kg soil dw (application via powder on feed)

≥ 20 mg/kg soil dw (application via dispersion on soil) (not determined for biomass of P25 since inconsistent concentration-effect)

≥ 20 mg/kg soil dw (application via dispersion on feed) (not determined for biomass of P25 since inconsistent concentration-effect)

Validity criteria fulfilled:
yes
Remarks:
reported by the author
Conclusions:
In a standard toxicity test according to OECD 222 (2004), the three different nano-sized TiO2 materials NM 105 (21 nm, rutile/anatase), NM 101 (8 nm, anatase) and NM 103 (20 nm, rutile, coated) did not reduce the number of offspring produced by Eisenia andrei, and the number of living adult earthworms, when these test materials were applied to the test system via powder or via dispersion on feed or soil. Depending on the test set up, the derived 56 d and 28 d-NOEC values ranged from ≥ 20 (dispersion) to ≥ 1000 mg/kg soil dw (powder; nominal). On the contrary, significant stimulation of reproduction was observed for some Ti2O (P25, NM101) with increasing TiO2 NP levels. However, the effect may be due to an improvement of the test soil.


Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, limit test, well documented
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
yes
Remarks:
four instead of eight TiO2 replicates were tested at 400 and 1000 mg/kg soil dw, controls were tested with eight replicates.
Principles of method if other than guideline:
All tests were perforemd under dark conditions. Chronic toxicity, limit test: four instead of eight TiO2 replicates were tested at 400 and 1000 mg/kg soil dw, controls were tested with eight replicates.
GLP compliance:
no
Remarks:
Earthworm reproduction test with NM 101: Raw data was documented and balances as well as pipettes were used in the style of GLP. The quality assurance unit was not present and did not check the report; the report was not stored in an archiv.
Analytical monitoring:
yes
Details on sampling:
SOIL SAMPLING (ACUTE TOXICITY TEST)
- Soil aliquots of the earthworm acute toxicity tests were sampled from each three replicates of the controls (0 mg TiO2 material/kg; n=3), of three TiO2 treatment groups (1000 mg TiO2 material/kg; n=3) at the end of the test (14 d).
- Sample storage conditions before analysis: Samples were stored in a fridge at -20 °C until analysis.

WORM SAMPLING (ACUTE TOXICITY TEST)
- Worms of these controls and treatment groups, which already had purged their guts for 24 h, were also frozen at -20 °C until analysis.
- Two worms per replicate were digested. In total, the worms of three replicates per treatment group were analyzed, resulting in six independently digested worms per treatment group.
Vehicle:
no
Details on preparation and application of test substrate:
PREPARATION OF TEST DISPERSION
- Direct addition of test material into test vessel: no
- Sonication of stock dispersion: stock suspensions were prepared by dispersing the TiO2 material in deionized water for 15 min by using an ultrasound probe (200 W, pulsed ultrasound: 0.2 s pulse and 0.8 s pause at 100% power, Sonoplus 200 W, BANDELIN Electronic GmbH & Co. KG). The preparation of the TiO2 dispersions occurred directly before they were applied to the test soil. To reach 400 and 1000 mg/kg soil dw, either 400 or 1000 mg TiO2 were applied to 160 ml deionized water.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Test medium: deionised water

APPLICATION OF TEST DISPERSION
- 160 ml of the test dispersions and a specific volume of deionized water were applied to a specific amount of dry test soil to reach TiO2 conentrations of 400 and 1000 mg/kg soil dw and a water capacitiy of 55% of the maximum water holding capacity.
- All constituents were mixed with a mixer for 5 min.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source:IBACON GmbH (Rossdorf, Germnay)
- Age at test initiation (mean and range, SD): adult, with clitellum
- Weight at test initiation (mean and range, SD): 250 - 600 mg ww

ACCLIMATION
- Acclimation period: 24 h
- Acclimation conditions (same as test or not): test condition (acute 20 °C and continous illumination; chronic: 20 °C and a light dark rhythm of 16:8 h)
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
yes
Total exposure duration:
28 d
Remarks:
acute: 14 d; chronic 28 d
Post exposure observation period:
cocoons were exposed to the test soil for further 28 d
Test temperature:
20 °C
pH:
test start: 4.7-5.0
test end: 5.5-6.3
Moisture:
water holding capacity:
test start: 46.3%-58.4%
test end: 46.6%-68.3%
Details on test conditions:
TEST SYSTEM
- Test container (material, size):test beaker
- Amount of soil or substrate: 500 g dw
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: acute and chronic toxicity test: 4
- No. of replicates per control: acute toxicity test: 4; chronic toxicity test: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Schmallenberg, Germany
- Soil taxonomic classification: RefeSol 01-A, slightly loamy, middle acidic, very slightly humic soil
- Organic carbon (%): 0.93
- Maximum water holding capacity (in % dry weigth): 29.8
- Pretreatment: soil was air dried, sieved through a 2 mm sieveand sotred at room temperature until use


OTHER TEST CONDITIONS
- Photoperiod
- acute toxcicity test: continuous light
- reproduction test: light/ dark rhythm 16:8 h
- Light intensity: 400 - 800 lux
- Food: acute test: no food; chronic test: dung of an organic cow farm, dried and ground to a fine powder
Nominal and measured concentrations:
nominal applied TiO2concentrations: acute test: 1000 mg TiO2/kg dw; chronic test: 400 and 1000 mg/kg soil dw
Control soils. ca. 2800 mg TiO2/kg dw
Soils applied with 1000 mg/kg of the TiO2: ca. 3800 mg/kg dw soil (recovery: 87-96%)
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Hombikat UV 100; PC 105
Basis for effect:
mortality
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Hombikat UV 100; PC 105
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Hombikat UV 100; PC 105
Basis for effect:
mortality
Details on results:
As recovery of TiO2 was > 80% of the nominal concentration, effect values are based on nominal concentrations.

Determined worm TiO2 concentrations are not valid, beacuse digestion was not complete. This was indicated by soil residues in the digested samples.

According to the OECD guideline 222 the coefficients of variation (CV) between the control replicates has to be <= 30%. This criteria was met in the tests with an application level of 400 mg NM 101/kg soil dw (13%) and 1000 mg NM 102/kg soil dw (9.4%). However, the CV in the tests with 1000 mg NM 101/kg soil dw and 400 mg NM 102/kg soil dw were above this limit (31.4% and 39.3%). Neverthless, the latter tests are considered valid, because all other validity criteria were met (controls: 30 juveniles per 10 adult worms, and adult mortality lower than 10% after 28 d of exposure) and the CV of the treatment replicates were lower than 30%, confirming the reliability of the results.

Reported statistics and error estimates:
Data were statistically analyzed with ToxRat® Professional (version 2.10, ToxRat solutions GmbH). Concentration response functions were fitted to the data using probit analysis. The concentrations causing 10 and 50% mortality (LC10 and 50) were calculated from this function. Significant differences to the control (*P<0.05) were determined using Fisher’s Exact Binominal Test with Bonferroni Correction to derive the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC).
Significant differences between the treatment groups and the controls were determined using student-t test for homogeneous variances or one way ANOVA (*P <0,05).
Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the nano-sized TiO2 materials Hombikat UV 100 (7-10 nm, anatase) and PC 105 (15-25 nm, anatase), which were applied to a natural test soil, did not affect survival of Eisenia fetida in an acute limit toxicity test (OECD 207) and did not affect growth and reproduction in an earthworm reproduction limit test (OECD 222), resulting in 28 d-NOEC values of ≥ 1000 mg/kg soil dw for survival and reproduction. Recovery of TiO2 was greater than 80% in the acute toxicity tests, allowing to base NOEC values on nominal concentrations.
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, limit test, well documented; all tests were performed under dark conditions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
2004
Deviations:
yes
Remarks:
imit test: four instead of eight TiO2 replicates were tested at 400 and 1000 mg/kg soil dw, controls were tested with eight replicates.
Principles of method if other than guideline:
Chronic toxicity, limit test: four instead of eight TiO2 replicates were tested at 400 and 1000 mg/kg soil dw, controls were tested with eight replicates.
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
yes
Details on sampling:
SOIL SAMPLING (ACUTE TOXICITY TEST)
- Soil aliquots of the earthworm acute toxicity tests were sampled from each three replicates of the controls (0 mg TiO2 material/kg; n=3), of three TiO2 treatment groups (1000 mg TiO2 material/kg; n=3) at the end of the test (14 d).
- Sample storage conditions before analysis: Samples were stored in a fridge at -20 °C until analysis.

WORM SAMPLING (ACUTE TOXICITY TEST)
- Worms of these controls and treatment groups, which already had purged their guts for 24 h, were also frozen at -20 °C until analysis.
- Two worms per replicate were digested. In total, the worms of three replicates per treatment group were analyzed, resulting in six independently digested worms per treatment group.
Vehicle:
no
Details on preparation and application of test substrate:
PREPARATION OF TEST DISPERSION
- Direct addition of test material into test vessel: no
- Sonication of stock dispersion: stock suspensions were prepared by dispersing the TiO2 material in deionized water for 15 min by using an ultrasound probe (200 W, pulsed ultrasound: 0.2 s pulse and 0.8 s pause at 100% power, Sonoplus 200 W, BANDELIN Electronic GmbH & Co. KG). The preparation of the TiO2 dispersions occurred directly before they were applied to the test soil. To reach 400 and 1000 mg/kg soil dw, either 400 or 1000 mg TiO2 were applied to 160 ml deionized water.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Test medium: deionised water

APPLICATION OF TEST DISPERSION
- 160 ml of the test dispersions and a specific volume of deionized water were applied to a specific amount of dry test soil to reach TiO2 conentrations of 400 and 1000 mg/kg soil dw and a water capacitiy of 55% of the maximum water holding capacity.
- All constituents were mixed with a mixer for 5 min.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source:IBACON GmbH (Rossdorf, Germnay)
- Age at test initiation (mean and range, SD): adult, with clitellum
- Weight at test initiation (mean and range, SD): 250 - 600 mg ww

ACCLIMATION
- Acclimation period: 24 h
- Acclimation conditions (same as test or not): test condition (acute 20 °C and continous illumination; chronic: 20 °C and a light dark rhythm of 16:8 h)
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
yes
Total exposure duration:
28 d
Remarks:
acute: 14 d; chronic 28 d
Post exposure observation period:
cocoons were exposed to the test soil for further 28 d
Test temperature:
20 °C
pH:
test start: 4.7-5.0
test end: 5.5-6.3
Moisture:
water holding capacity:
test start: 46.3%-58.4%
test end: 46.6%-68.3%
Details on test conditions:
TEST SYSTEM
- Test container (material, size):test beaker
- Amount of soil or substrate: 500 g dw
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: acute and chronic toxicity test: 4
- No. of replicates per control: acute toxicity test: 4; chronic toxicity test: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Schmallenberg, Germany
- Soil taxonomic classification: RefeSol 01-A, slightly loamy, middle acidic, very slightly humic soil
- Organic carbon (%): 0.93
- Maximum water holding capacity (in % dry weigth): 29.8
- Pretreatment: soil was air dried, sieved through a 2 mm sieveand sotred at room temperature until use


OTHER TEST CONDITIONS
- Photoperiod
- acute toxcicity test: continuous light
- reproduction test: light/ dark rhythm 16:8 h
- Light intensity: 400 - 800 lux
- Food: acute test: no food; chronic test: dung of an organic cow farm, dried and ground to a fine powder
Nominal and measured concentrations:
nominal applied TiO2concentrations: acute test: 1000 mg TiO2/kg dw; chronic test: 400 and 1000 mg/kg soil dw
Control soils. ca. 2800 mg TiO2/kg dw
Soils applied with 1000 mg/kg of the TiO2: ca. 3800 mg/kg dw soil (recovery: 87-96%)
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tiona AT1
Basis for effect:
mortality
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tiona AT1
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tiona AT1
Basis for effect:
mortality
Details on results:
As recovery of TiO2 was > 80% of the nominal concentration, effect values are based on nominal concentrations.

Determined worm TiO2 concentrations are not valid, beacuse digestion was not complete. This was indicated by soil residues in the digested samples.

According to the OECD guideline 222 the coefficients of variation (CV) between the control replicates has to be <= 30%. The CV in the tests with 400 and 1000 mg NM 100/kg soil dw were above this limit (31.4% and 39.3%). Neverthless, the latter tests are considered valid, because all other validity criteria were met (controls: 30 juveniles per 10 adult worms, and adult mortality lower than 10% after 28 d of exposure) and the CV of the treatment replicates were lower than 30%, confirming the reliability of the results.

Reported statistics and error estimates:
Data were statistically analyzed with ToxRat® Professional (version 2.10, ToxRat solutions GmbH). Concentration response functions were fitted to the data using probit analysis. The concentrations causing 10 and 50% mortality (LC10 and 50) were calculated from this function. Significant differences to the control (*P<0.05) were determined using Fisher’s Exact Binominal Test with Bonferroni Correction to derive the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC).
Significant differences between the treatment groups and the controls were determined using student-t test for homogeneous variances or one way ANOVA (*P <0,05).
Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the macro-sized TiO2 material Tiona AT1 (200 nm, anatase), which were applied to a natural test soil, did not affect survival of Eisenia fetida in an acute limit toxicity test (OECD 207) and did not affect growth and reproduction in an earthworm reproduction limit test (OECD 222), resulting in 28 d-NOEC values of ≥ 1000 mg/kg soil dw for survival and reproduction. Recovery of TiO2 was greater than 80% in the acute toxicity tests, allowing to base NOEC values on nominal concentrations.

Description of key information

 Several studies are available and indicate that nano- and microsized TiO2 (including anatase, rutile and mixed forms) in natural soil and artificial substrate do not affect survival, reproduction and growth of Eisenia fetida and Eisenia andrei up to at least 1000 mg/kg.

Key value for chemical safety assessment

Additional information

Regarding toxicity to the reproduction of Eisenia fetida and Eisenia andrei, Schlich et al. (2012) and Wyrwoll et al. (2014) did not observe effects of nanosized and microsized TiO2 up to 1000 mg/kg dw soil:


In the study by Wyrwoll et al. (2014), dispersions of the microsized TiO2 material Tiona AT1 (200 nm, anatase), and of the nano-sized TiO2 materials Hombikat UV 100 (7-10 nm, anatase) and PC 105 (15-25 nm, anatase) in a natural test soil did not affect the survival of Eisenia fetida in the acute limit toxicity test (OECD 207) and did not affect growth and reproduction in the earthworm reproduction limit test (OECD 222) resulting in 28-d NOEC values of ≥ 1000 mg/kg soil dw (nominal) for survival and reproduction. Schlich et al. (2012) tested three different nano-sized TiO2 materials, i.e. NM 105 (21 nm, rutile/anatase), NM 101 (8 nm, anatase) and NM 103 (20 nm, rutile, coated), in a standard toxicity test according to OECD 222 (2004). The TiO2 materials did not reduce the number of offspring, and adults of Eisenia andrei when applied to the test system via powder or dispersion on feed or soil. Depending on the test setup, the 56-d and 28-d NOEC values ranged from ≥ 20 (dispersion) to ≥ 1000 mg/kg soil dw (powder; nominal). On the contrary, significant stimulation of reproduction was observed for some TiO2 materials (P25, NM 101) with increasing levels. However, the effect may be due to an improvement of the test soil.

Several supporting studies on the toxicity of nanosized and microsized TiO2 to earthworm exist:

Micro-TiO2 material Titanium oxide-325 mesh (< 40 µm) did not affect survival and reproduction of Eisenia andrei and survival, reproduction and juvenile growth of Eisenia fetida in a sandy-loam soil resulting in 28-d, 56-d and 126 d NOEC values of ≥ 200 mg/kg dw (nominal) in a study by McShane et al. (2012). Higher concentrations were applied to an artificial substrate and the natural soil by dry-mixing resulting in 28-d and 56-d NOEC values of ≥ 10,000 mg/kg soil dw (nominal) for all endpoints. The toxicity of nanosized TiO2 material to the survival and reproduction of Eisenia andrei and Eisenia fetida was studied in artificial substrate and in a natural soil, the latter only with E. Andrei, according to OECD 207 and OECD 222, resulting in 28-d and 56-d NOEC values of ≥ 10,000 mg/kg soil dw (nominal) for adult survival and reproduction. However, tests were performed in the dark to avoid potential phototoxicity and thus potentially reducing the exposure of the worms. Under standard-light-darkness cycles, earthworms tend to remain in the soil under light conditions. Hence, results of these experiments may not be conservative and are considered supporting data.

Further, McShane et al. (2012) observed in an earthworm avoidance test with Eisenia fetida that the avoidance behaviour was not affected by microsized TiO2 at 10,000 mg/kg soil. Results for the three nano-sized TiO2 materials vary but indicate that earthworms do not avoid TiO2-NP concentrations up to 100 mg/kg soil but consistently avoid higher levels of 10,000 mg/kg soil.

Whitfield Aslund et al. (2012) used metabolomics to examine the response of Eisenia fetida earthworms raised from juveniles for 20−23 weeks in soil spiked with 20 or 200 mg/kg nano- and microsized TiO2. Data seem to indicate that earthworms exhibited changes in their metabolic profile relative to the control after exposure to 200 mg/kg of nano- and microsized TiO2. However, the correlation between the observed metabolomic response and ecologically relevant endpoints is missing to date.

Lapied, E. 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 up to 100 mg/kg. Apoptosis was measured by immunohistochemistry. Mortality and apoptotic frequency were not affected in soil-exposed worms. Based on Ti localization by XRF microscopy, nanoparticles appear to be rather inert and did not cross the intestinal epithelium/ chloragogenous matrix barrier to enter the coelomic liquid, or the cuticular barrier to reach the muscular layers.

The fact that direct exposure to TiO2 NPs did not impair the immuno-effectiveness of earthworm Eisenia veneta (Rosa) coelomocyte cells at concentrations well above those predicted for sewage sludge in the UK (25 mg/L) appears to be further evidence of the low potential of TiO2 nanoparticles for toxicity to earthworms (Johnson et al. 2011).

In sum and applying a weight of evidence approach, nano- and microsized TiO2 (rutile, anatase and mixed forms) appears to have a low potential for short- and long-term toxicity to soil annelides based on data available for different earthworm species up to at least 1000 mg/kg dw soil.