Titanium carbide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Meets generally accepted scinetific standards, well documented and acceptable for assessment. Purity and batch of test metarial is not reported, however, as the authors did derive no-effect concentrations potantial impurities did not adversily affect the test results. Justification for read-across: Due to similar or lower transformation/dissolution results for titanium carbide (the target substance) than titanium dioxide (the source substance), the resulting toxicity potential would also be expected to be similar or lower, so read-across is appropriate. Therefore, the dose descriptors are expected to be sufficiently similar or higher for the target substance, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was conducted to determine the toxic effects of nanoscale TiO2 (small size: 66 nm, medium size: 950 nm , large size: 44µm) to Gram-positive bacteria Bacillus subtilis. Objectives of the study were to:
- determine toxic effects concentrations
- evaluate whether toxicity is related to particle size
- evaluate whether natural sunlight produces enough reactive oxygen species (ROS) to stimulate TiO2 toxicity (experiment with small size particles only at pre-determined toxic concentrations)

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
* TiO2 powder was added to 100 ml Milli-Q s water to obtain a 10 g/L soultiuon;
* solution was shaken vigorously

Test organisms (species):

Bacillus subtilis

Details on inoculum:

- Laboratory culture: Maintenance on Luria-Bertani (LB) plates
- Method of cultivation: cultivation in minimal Davis Medium (MD) with reduced potassium phosphate compared to the original Davis medium in order to avoid precipitation of nanoscale test material due to high phosphate concentrations

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 h

Remarks on exposure duration:

Total incubation period: 14-20 h

Post exposure observation period:

Not applicable.

Hardness:

Not applicable.

Test temperature:

36 °C

pH:

7.0

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentrations: 0 (control: medium and bacteria only), 10, 50, 100, 500, 1000, 2000, and 5000 ppm

Details on test conditions:

TEST SYSTEM
- Test vessel: Petri dishes
- Type (delete if not applicable): closed (sealed with parafilm (American National Can, Chicago, IL, USA)) and wrapped with aluminium foil to avoid illumination if required
- Fill volume: 5 ml nanoparticle suspension
- Aeration: not applicable
- Plates were placed on a rocker platform (Bell Company Biotechnology, Vineland, NJ, USA) to maintain particles in suspension
- Initial cell concentration: ca. 10³ CFU/ml
- No. of vessels per concentration (replicates): 6


TEST MEDIUM / WATER PARAMETERS (minimal Davis medium):
- Ingredients in 1 L Milli-Q water:
* K2HPO4: 0.7 g
* KH2PO4: 0.2 g
* (NH4)2SO4: 1 g
* Na-citrate: 0.5 g
* MgSO4 * 7H2O: 0.1 g
* Glucose: 1 g

OTHER TEST CONDITIONS (in experiments to determine influence of illumination)
- Photoperiod: 6 h (9 AM to 3 PM; bright October day, Houston, Texas)
- Light intensity: direct natural sunlight (window of a laboratory facing southeast), UV Index 6-7; average outdoor incident luminescence: 50.4 klux/h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth inhibition

Reference substance (positive control):

no

Key result

Duration:

14 h

Dose descriptor:

NOEC

Effect conc.:

500 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC75

Effect conc.:

1 000 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks on result:

other: standard deviation: ± 6.6 %; illumination of samples with natural sunlight

Details on results:

- True particle size distribution: significantly different from advertised particle sizes (see Table 1 for details) probably due to aggregation of particles:
=> similar particle size for all three nanomaterials used
=> no effect of advertised particle size on toxicity
- Illumination significantly (p < 0.05) enhanced toxicity (see Fig. 2 below): growth inhibition was 2.5-fold greater in the presence of light

Results with reference substance (positive control):

Not applicable.

Table 1: Measurement of particle size ranges and mean size for all suspensions

Suspension	Terminology	Advertised particle size (nm)	Actual particle size range in suspension (nm)	Actual mean particle size in suspension (nm)
TiO2	Small	66	175-810	330
	Medium	950	240-460	320
	Large	44,000	1000	1000

 

Validity criteria fulfilled:

not applicable

Conclusions:

The 14-h NOEC for TiO2 (330 nm) to Bacillus subtilis was determined to be 500 ppm. The next higher concentration of 1000 ppm caused 75 % inhibition when samples were illuminated by natural sunlight.
Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to Bacillus subtilis. Growth inhibition was 2.5-fold greater in the presence of light. Thus, the authors conclude that the antibacterial toxicity of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.

Executive summary:

This study was conducted to determine the toxic effects of nanoscale TiO2 (advertised particle sizes: "small size": 66 nm, "medium size": 950 nm , "large size": 44µm) to Gram-positive bacteria Bacillus subtilis. Objectives of the study were to:

- determine toxic effects concentrations

- evaluate whether toxicity is related to particle size

- evaluate whether natural sunlight produces enough reactive oxygen species (ROS) to stimulate TiO2 toxicity (experiments with "small size" particles only at pre-determined toxic concentrations).

The 14-h NOEC for TiO2 (330 nm) to Bacillus subtilis was determined to be 500 ppm. The next higher concentration of 1000 ppm caused 75 % inhibition when samples were illuminated by natural sunlight. Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to Bacillus subtilis. Growth inhibition was 2.5-fold greater in the presence of light.

Thus, the authors conclude that the antibacterial toxicity of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.

The true particle size distribution was significantly different from advertised particle sizes probably due to aggregation of particles. The authors determined similar particle sizes for all three nanomaterials used (mean particle size for "small" particles, "medium" particles and "large" particles were 330 nm, 320 nm, and 1000 nm, respectively). Therefore, no effects of the advertised particle size on toxicity to bacteria could be determined.

Reference 2

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Meets generally accepted scinetific standards, well documented and acceptable for assessment. Purity and batch of test metarial is not reported, however, as the authors did derive no-effect concentrations potantial impurities did not adversily affect the test results. Justification for read-across: Due to similar or lower transformation/dissolution results for titanium carbide (the target substance) than titanium dioxide (the source substance), the resulting toxicity potential would also be expected to be similar or lower, so read-across is appropriate. Therefore, the dose descriptors are expected to be sufficiently similar or higher for the target substance, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was conducted to determine the toxic effects of nanoscale TiO2 (small size: 66 nm, medium size: 950 nm , large size: 44µm) to Gram-negative bacteria E. coli. Objectives of the study were to:
- determine toxic effects concentrations
- evaluate whether toxicity is related to particle size
- evaluate whether natural sunlight produces enough reactive oxygen species (ROS) to stimulate TiO2 toxicity (experiment with small size particles only at pre-determined toxic concentrations)

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
* TiO2 powder was added to 100 ml Milli-Q s water to obtain a 10 g/L soultiuon;
* solution was shaken vigorously

Test organisms (species):

Bacillus subtilis

Details on inoculum:

- Laboratory culture: Maintenance on Luria-Bertani (LB) plates
- Method of cultivation: cultivation in minimal Davis Medium (MD) with reduced potassium phosphate compared to the original Davis medium in order to avoid precipitation of nanoscale test material due to high phosphate concentrations

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 h

Remarks on exposure duration:

Total incubation period: 14-20 h

Post exposure observation period:

Not applicable.

Hardness:

Not applicable.

Test temperature:

36 °C

pH:

7.0

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentrations: 0 (control: medium and bacteria only), 10, 50, 100, 500, 1000, 2000, and 5000 ppm

Details on test conditions:

TEST SYSTEM
- Test vessel: Petri dishes
- Type (delete if not applicable): closed (sealed with parafilm (American National Can, Chicago, IL, USA)) and wrapped with aluminium foil to avoid illumination if required
- Fill volume: 5 ml nanoparticle suspension
- Aeration: not applicable
- Plates were placed on a rocker platform (Bell Company Biotechnology, Vineland, NJ, USA) to maintain particles in suspension
- Initial cell concentration: ca. 10³ CFU/ml
- No. of vessels per concentration (replicates): 6


TEST MEDIUM / WATER PARAMETERS (minimal Davis medium):
- Ingredients in 1 L Milli-Q water:
* K2HPO4: 0.7 g
* KH2PO4: 0.2 g
* (NH4)2SO4: 1 g
* Na-citrate: 0.5 g
* MgSO4 * 7H2O: 0.1 g
* Glucose: 1 g

OTHER TEST CONDITIONS (in experiments to determine influence of illumination)
- Photoperiod: 6 h (9 AM to 3 PM; bright October day, Houston, Texas)
- Light intensity: direct natural sunlight (window of a laboratory facing southeast), UV Index 6-7; average outdoor incident luminescence: 50.4 klux/h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth inhibition

Reference substance (positive control):

no

Key result

Duration:

14 h

Dose descriptor:

NOEC

Effect conc.:

100 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC15

Effect conc.:

500 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: standard deviation: ± 4.2 %; illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC44

Effect conc.:

1 000 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: standard deviation: ± 7.0 %; illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC46

Effect conc.:

2 000

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: standard deviation: ± 11.3 %; illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC72

Effect conc.:

5 000 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: standard deviation: ± 9.4 %; illumination of samples with natural sunlight

Details on results:

- True particle size distribution: significantly different from advertised particle sizes (see Table 1 for details) probably due to aggregation of particles:
=> similar particle size for all three nanomaterials used
=> no effect of advertised particle size on toxicity
- Illumination significantly (p < 0.05) enhanced toxicity (see Fig. 2 below): growth inhibition was 1.8-fold greater in the presence of light

Results with reference substance (positive control):

Not applicable.

Table 1: Measurement of particle size ranges and mean size for all suspensions

Suspension	Terminology	Advertised particle size (nm)	Actual particle size range in suspension (nm)	Actual mean particle size in suspension (nm)
TiO2	Small	66	175-810	330
	Medium	950	240-460	320
	Large	44,000	1000	1000

 

Validity criteria fulfilled:

not applicable

Conclusions:

The 14-h NOEC for TiO2 (330 nm) to Bacillus subtilis was determined to be 100 ppm. 44±7.0 % and 46±11.3 % inhibition could be observed at 1000 ppm and 2000 ppm, respectively, in samples illuminated by natural sunlight.
Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to E.coli. Growth inhibition was 1.8-fold greater in the presence of light. Thus, the authors conclude that the antibacterial toxicity of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.

Executive summary:

This study was conducted to determine the toxic effects of nanoscale TiO2 (advertised particle sizes: "small size": 66 nm, "medium size": 950 nm , "large size": 44 µm) to Gram-negative bacteria E. coli. Objectives of the study were to:

- determine toxic effects concentrations

- evaluate whether toxicity is related to particle size

- evaluate whether natural sunlight produces enough reactive oxygen species (ROS) to stimulate TiO2 toxicity (experiments with "small size" particles only at pre-determined toxic concentrations).

The 14-h NOEC for TiO2 (330 nm) to E.coli was determined to be 100 ppm. 44±7.0 % and 46±11.3 % inhibition could be observed at 1000 ppm and 2000 ppm, respectively, in samples illuminated by natural sunlight. Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to E.coli. Growth inhibition was 1.8-fold greater in the presence of light.

Thus, the authors conclude that the antibacterial toxicity of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.

The true particle size distribution was significantly different from advertised particle sizes probably due to aggregation of particles. The authors determined similar particle sizes for all three nanomaterials used (mean particle size for "small" particles, "medium" particles and "large" particles were 330 nm, 320 nm, and 1000 nm, respectively). Therefore, no effects of the advertised particle size on toxicity to bacteria could be determined.

Reference 3

Endpoint:

toxicity to microorganisms, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Justification for read-across: Due to lower transformation/dissolution results for titanium carbide (the target substance) than titanium dioxide (the source substance), the resulting toxicity potential would also be expected to be lower, so read-across is appropriate. Therefore, the dose descriptors are expected to be sufficiently higher for the target substance, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach (see IUCLID section 13.2).

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to same study

Key result

Duration:

14 h

Dose descriptor:

NOEC

Effect conc.:

500 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC75

Effect conc.:

1 000 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks on result:

other: standard deviation: ± 6.6 %; illumination of samples with natural sunlight

Details on results:

- True particle size distribution: significantly different from advertised particle sizes (see Table 1 for details) probably due to aggregation of particles:
=> similar particle size for all three nanomaterials used
=> no effect of advertised particle size on toxicity
- Illumination significantly (p < 0.05) enhanced toxicity (see Fig. 2 below): growth inhibition was 2.5-fold greater in the presence of light

Results with reference substance (positive control):

Not applicable.

Table 1: Measurement of particle size ranges and mean size for all suspensions

Suspension	Terminology	Advertised particle size (nm)	Actual particle size range in suspension (nm)	Actual mean particle size in suspension (nm)
TiO2	Small	66	175-810	330
	Medium	950	240-460	320
	Large	44,000	1000	1000

 

Validity criteria fulfilled:

not applicable

Conclusions:

The 14-h NOEC for TiO2 (330 nm) to Bacillus subtilis was determined to be 500 ppm. The next higher concentration of 1000 ppm caused 75 % inhibition when samples were illuminated by natural sunlight.
Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to Bacillus subtilis. Growth inhibition was 2.5-fold greater in the presence of light. Thus, the authors conclude that the antibacterial toxicity of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.

Executive summary:

This study was conducted to determine the toxic effects of nanoscale TiO2 (advertised particle sizes: "small size": 66 nm, "medium size": 950 nm , "large size": 44µm) to Gram-positive bacteria Bacillus subtilis. Objectives of the study were to:

- determine toxic effects concentrations

- evaluate whether toxicity is related to particle size

- evaluate whether natural sunlight produces enough reactive oxygen species (ROS) to stimulate TiO2 toxicity (experiments with "small size" particles only at pre-determined toxic concentrations).

 

The 14-h NOEC for TiO2 (330 nm) to Bacillus subtilis was determined to be 500 ppm. The next higher concentration of 1000 ppm caused 75 % inhibition when samples were illuminated by natural sunlight. Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to Bacillus subtilis. Growth inhibition was 2.5-fold greater in the presence of light.

Thus, the authors conclude that the antibacterial toxicity of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.

 

The true particle size distribution was significantly different from advertised particle sizes probably due to aggregation of particles. The authors determined similar particle sizes for all three nanomaterials used (mean particle size for "small" particles, "medium" particles and "large" particles were 330 nm, 320 nm, and 1000 nm, respectively). Therefore, no effects of the advertised particle size on toxicity to bacteria could be determined.

This information is used in a read-across aproach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 4

Endpoint:

toxicity to microorganisms, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to same study

Key result

Duration:

14 h

Dose descriptor:

NOEC

Effect conc.:

100 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC15

Effect conc.:

500 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: standard deviation: ± 4.2 %; illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC44

Effect conc.:

1 000 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: standard deviation: ± 7.0 %; illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC46

Effect conc.:

2 000

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: standard deviation: ± 11.3 %; illumination of samples with natural sunlight

Duration:

14 h

Dose descriptor:

other: EC72

Effect conc.:

5 000 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

TiO2 (330 nm)

Basis for effect:

growth inhibition

Remarks:

in %

Remarks on result:

other: standard deviation: ± 9.4 %; illumination of samples with natural sunlight

Details on results:

- True particle size distribution: significantly different from advertised particle sizes (see Table 1 for details) probably due to aggregation of particles:
=> similar particle size for all three nanomaterials used
=> no effect of advertised particle size on toxicity
- Illumination significantly (p < 0.05) enhanced toxicity (see Fig. 2 below): growth inhibition was 1.8-fold greater in the presence of light

Results with reference substance (positive control):

Not applicable.

Table 1: Measurement of particle size ranges and mean size for all suspensions

Suspension	Terminology	Advertised particle size (nm)	Actual particle size range in suspension (nm)	Actual mean particle size in suspension (nm)
TiO2	Small	66	175-810	330
	Medium	950	240-460	320
	Large	44,000	1000	1000

 

Validity criteria fulfilled:

not applicable

Conclusions:

The 14-h NOEC for TiO2 (330 nm) to Bacillus subtilis was determined to be 100 ppm. 44±7.0 % and 46±11.3 % inhibition could be observed at 1000 ppm and 2000 ppm, respectively, in samples illuminated by natural sunlight.
Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to E.coli. Growth inhibition was 1.8-fold greater in the presence of light. Thus, the authors conclude that the antibacterial toxicity of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.

Executive summary:

This study was conducted to determine the toxic effects of nanoscale TiO2 (advertised particle sizes: "small size": 66 nm, "medium size": 950 nm , "large size": 44 µm) to Gram-negative bacteria E. coli. Objectives of the study were to:

- determine toxic effects concentrations

- evaluate whether toxicity is related to particle size

- evaluate whether natural sunlight produces enough reactive oxygen species (ROS) to stimulate TiO2 toxicity (experiments with "small size" particles only at pre-determined toxic concentrations).

 

The 14-h NOEC for TiO2 (330 nm) to E.coli was determined to be 100 ppm. 44±7.0 % and 46±11.3 % inhibition could be observed at 1000 ppm and 2000 ppm, respectively, in samples illuminated by natural sunlight. Illumination significantly (p < 0.05) enhanced toxicity of TiO2 to E.coli. Growth inhibition was 1.8-fold greater in the presence of light.

Thus, the authors conclude that the antibacterial toxicity of TiO2 is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO2 toxicity also occurs under dark conditions.

 

The true particle size distribution was significantly different from advertised particle sizes probably due to aggregation of particles. The authors determined similar particle sizes for all three nanomaterials used (mean particle size for "small" particles, "medium" particles and "large" particles were 330 nm, 320 nm, and 1000 nm, respectively). Therefore, no effects of the advertised particle size on toxicity to bacteria could be determined.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13). 

Reference 5

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Documentation insufficient for assessment. Justification for read-across: Due to similar or lower transformation/dissolution results for titanium carbide (the target substance) than titanium dioxide (the source substance), the resulting toxicity potential would also be expected to be similar or lower, so read-across is appropriate. Therefore, the dose descriptors are expected to be sufficiently similar or higher for the target substance, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Flash assay (> kinetic luminescence inhibition test taking into account the color and turbidity of the sample) and subsequent growth inhibition measurements with Vibrio fischeri on agar.

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

tock solutions of 40 g/L were prepared in Milli-Q water and were sonicated for 30 min. Solutions were stored in the dark at +4 °C. Before toxicity testing, stocks were vortexed

Test organisms (species):

Vibrio fisheri

Details on inoculum:

- Laboratory culture: not applicable; Vibrio fischeri reagent (V. fischeri NRRL-B 11177 from Aboatox, Turku, FInland) was used
- Bacterial suspensions (concentrated) in 10 % glycerol were stored at -80 °C and thawed for use

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Hardness:

Not applicable.

Test temperature:

20 °C

pH:

Not reported.

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal total concentrations: up to 20,000 mg/L (no further details reported).

Details on test conditions:

TEST SYSTEM
- Test vessel: test cuvettes (luminescence test)
- No. of organisms per vessel: 10^6 CFU/ml (in 2 % NaCl); CFU were quantified in agar medium (see below)
- No. of vessels per concentration (replicates): 3


AGAR MEDIUM (ingredients per L):
- NaCl: 30 g
- Na2HPO4 * 2H2O: 4.7 g
- KH2PO4: 1 g
- (NH4)2HPO4: 0.5 g
- MgSO4 * 7H2O: 0.3 g
- Tryptone (Bacto): 5 g
- Yeat extract (Bacto): 3 g
- Glycerol (99 %): 2 ml
- Agar: 15 g


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): luminescence and growth inhibition

Reference substance (positive control):

no

Key result

Duration:

30 min

Dose descriptor:

NOEC

Effect conc.:

> 20 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: luminescence

Duration:

8 h

Dose descriptor:

other: MIC

Effect conc.:

> 20 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

No further details reported.

Reported statistics and error estimates:

Inhibition [%] as well as EC50 and EC20 values were calculated by Biotox software (Thermo Labsystems, Helsinki, Finland).

Validity criteria fulfilled:

not applicable

Conclusions:

TiO2 is not inhibitory for growth and luminescence of V. fischeri up to concentrations of 20,000 mg/L.

Executive summary:

The toxicity of TiO2 nanoparticles to Vibrio fischeri was investigated by measurements of inhibition of luminescence (> Flash Assay) and growth.

The Flash Assay is a luminescence inhibition test that takes into account sample color and turbidity. Measurements were performed with a 1251 Luminometer (Thermo Labsystems, Helsinki, Finland). Luminescence is measured every 0.05 s under continuous mixing at ambient temperature (20 °C). All chemicals were diluted and tested in isotonic solution for V. fischeri (2 % NaCl) and inhibition of the bacterial luminescence was calculated [%] after 30 min. NOEC (defined as < 20 % inhibition), EC20 and EC50 values were calculated.

After the Flash Assay was completed the test cuvettes were further incubated for 7 h at +15 °C in the dark. Subsequently, suspensions were vortexed and 10 µl of suspension was streaked on agar medium. Bacteria were then grown for 72 h (20 °C, incubation in the dark) and luminescent colonies of the bacteria V. fischeri were visually checked in the dark to determine the minimal Inhibitory Concentration (MIC = lowest test concentration used for preincubation of V. fischeri during 8 h that causes total inhibition of growth on agar media after growing for 72 h).

Under the conditions of this study TiO2 is not inhibitory for growth and luminescence of V. fischeri up to concentrations of 20,000 mg/L.

Reference 6

Endpoint:

toxicity to microorganisms, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

30 min

Dose descriptor:

NOEC

Effect conc.:

> 20 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: luminescence

Duration:

8 h

Dose descriptor:

other: MIC

Effect conc.:

> 20 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

No further details reported.

Reported statistics and error estimates:

Inhibition [%] as well as EC50 and EC20 values were calculated by Biotox software (Thermo Labsystems, Helsinki, Finland).

Validity criteria fulfilled:

not applicable

Conclusions:

TiO2 is not inhibitory for growth and luminescence of V. fischeri up to concentrations of 20,000 mg/L.

Executive summary:

The toxicity of TiO2 nanoparticles to Vibrio fischeri was investigated by measurements of inhibition of luminescence (> Flash Assay) and growth.

 

The Flash Assay is a luminescence inhibition test that takes into account sample color and turbidity. Measurements were performed with a 1251 Luminometer (Thermo Labsystems, Helsinki, Finland). Luminescence is measured every 0.05 s under continuous mixing at ambient temperature (20 °C). All chemicals were diluted and tested in isotonic solution for V. fischeri (2 % NaCl) and inhibition of the bacterial luminescence was calculated [%] after 30 min. NOEC (defined as < 20 % inhibition), EC20 and EC50 values were calculated.

 

After the Flash Assay was completed the test cuvettes were further incubated for 7 h at +15 °C in the dark. Subsequently, suspensions were vortexed and 10 µl of suspension was streaked on agar medium. Bacteria were then grown for 72 h (20 °C, incubation in the dark) and luminescent colonies of the bacteria V. fischeri were visually checked in the dark to determine the minimal Inhibitory Concentration (MIC = lowest test concentration used for preincubation of V. fischeri during 8 h that causes total inhibition of growth on agar media after growing for 72 h).

 

Under the conditions of this study TiO2 is not inhibitory for growth and luminescence of V. fischeri up to concentrations of 20,000 mg/L.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached report (see IUCLID section 13).

Reference 7

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Documentation insufficient for assessment. Justification for read-across: Due to similar or lower transformation/dissolution results for titanium carbide (the target substance) than titanium tetrachloride (the source substance), the resulting toxicity potential would also be expected to be similar or lower, so read-across is appropriate. Therefore, the dose descriptors are expected to be sufficiently similar or higher for the target substance, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The toxicity of TiCl4 to Tetrahymena pyriformis was determined by using the flask technique. Samples with T. pyriformis in the exponential growth phase were incubated at least in triplicate for 9 hours with the test substance and growth inhibition of Tetrahymena pyrifoemis was determined by measuring the cellular density with a Coulter Counter.

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution was prepared in de-ionized water

Test organisms (species):

Tetrahymena pyriformis

Details on inoculum:

- Laboratory culture: axenic growth at 28 °C in PPYS-defined medium (contains proteose peptone and yeast extract (both Difco), as well as inorganic salts (Plessner et al. 1964)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 h

Hardness:

Not applicable.

Test temperature:

No data.

pH:

No data.

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal: five-step graded concentration series

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml Fernbach flasks
- Material, size, headspace, fill volume: 100 ml
- Cell density at time of dosing: 10^4 cells /ml
- No. of vessels per concentration (replicates): 3-5

TEST MEDIUM: PPYS

SAMPLING:
- Withdrawal of 1 ml aliquots directly after dosing and every hour for 9 hours.
- Samples were fixed in 1 ml of 4 % formaldehyde in Isoton buffer (Coultronics)
- Cellular density was determined by an electronic particles Coulter Counter ZM (Coultronics)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth (> increase of relative doubling time caused by the test substance)

Reference substance (positive control):

no

Key result

Duration:

9 h

Dose descriptor:

IC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Ti

Basis for effect:

growth inhibition

Details on results:

NO further details reported.

Results with reference substance (positive control):

Not applicable.

Validity criteria fulfilled:

not applicable

Conclusions:

Growth of Tetrahymena pyriformis is inhibited to 50 % by 20 mg Ti/L (applied as TiCl4).

Executive summary:

The toxicity of TiCl4 to Tetrahymena pyriformis was determined by using the flask technique. Samples with T. pyriformis in the exponential growth phase were incubated at least in triplicate for 9 hours with the test substance and growth inhibition of Tetrahymena pyriformis was determined by measuring the cellular density with a Coulter Counter.

Growth of Tetrahymena pyriformis is inhibited to 50 % by 20 mg Ti/L (applied as TiCl4).

Reference 8

Endpoint:

toxicity to microorganisms, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Key result

Duration:

9 h

Dose descriptor:

IC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Ti

Basis for effect:

growth inhibition

Details on results:

NO further details reported.

Results with reference substance (positive control):

Not applicable.

Validity criteria fulfilled:

not applicable

Conclusions:

Growth of Tetrahymena pyriformis is inhibited to 50 % by 20 mg Ti/L (applied as TiCl4).

Executive summary:

The toxicity of TiCl4 to Tetrahymena pyriformis was determined by using the flask technique. Samples with T. pyriformis in the exponential growth phase were incubated at least in triplicate for 9 hours with the test substance and growth inhibition of Tetrahymena pyriformis was determined by measuring the cellular density with a Coulter Counter.

Growth of Tetrahymena pyriformis is inhibited to 50 % by 20 mg Ti/L (applied as TiCl4).

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 9

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Documentation insufficient for assessment. Justification for read-across: Due to similar or lower transformation/dissolution results for titanium carbide (the target substance) than titanium tetrachloride(the source substance), the resulting toxicity potential would also be expected to be similar or lower, so read-across is appropriate. Therefore, the dose descriptors are expected to be sufficiently similar or higher for the target substance, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The toxicity of TiCl4 to Tetrahymena pyriformis was determined by using the flask technique. Samples with T. pyriformis in the exponential growth phase were incubated at least in triplicate for 9 hours with the test substance and growth inhibition of Tetrahymena pyrifoemis was determined by measuring the cellular density with a Coulter Counter. IC50 values were determined after 3, 6, and 9h, which was the time needed to get 1, 2, and 3 generations of T. pyriformis, respectively, in the control. .

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution was prepared in de-ionized water

Test organisms (species):

Tetrahymena pyriformis

Details on inoculum:

- Laboratory culture: axenic growth at 28 °C in PPYS-defined medium (contains proteose peptone and yeast extract (both Difco), as well as inorganic salts (Plessner et al. 1964)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 h

Hardness:

Not applicable.

Test temperature:

No data.

pH:

No data.

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

At least five concentration steps; no further details reported.

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 100 ml
- Cell density at time of dosing: 10^4 cells /ml
- No. of vessels per concentration (replicates): 3-5

TEST MEDIUM: PPYS

SAMPLING:
- Withdrawal of 1 ml aliquots directly after dosing and every hour for 9 hours.
- Samples were fixed in 1 ml of 4 % formaldehyde in Isoton buffer (Coultronics)
- Cellular density was determined by an electronic particles Coulter Counter ZM (Coultronics)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth

Reference substance (positive control):

no

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Ti

Basis for effect:

growth inhibition

Duration:

6 h

Dose descriptor:

IC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Ti

Basis for effect:

growth inhibition

Key result

Duration:

9 h

Dose descriptor:

IC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Ti

Basis for effect:

growth inhibition

Details on results:

No further details reported.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Regression analysis computed with Statview (v.2)

Validity criteria fulfilled:

not applicable

Conclusions:

Growth of Tetrahymena pyriformis is inhibited to 50 % by 35, 20, and 20 mg Ti/L after 3, 6, and 9 h, respectively.

Executive summary:

The toxicity of Ti to Tetrahymena pyriformis was determined by using the flask technique. Samples with T. pyriformis in the exponential growth phase were incubated at least in triplicate for 9 hours with the test substance and growth inhibition of Tetrahymena pyrifoemis was determined by measuring the cellular density with a Coulter Counter.

Growth of Tetrahymena pyriformis is inhibited to 50 % by 35, 20, and 20 mg Ti/L after 3, 6, and 9 h, respectively.

Reference 10

Endpoint:

toxicity to microorganisms, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Ti

Basis for effect:

growth inhibition

Duration:

6 h

Dose descriptor:

IC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Ti

Basis for effect:

growth inhibition

Key result

Duration:

9 h

Dose descriptor:

IC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Ti

Basis for effect:

growth inhibition

Details on results:

No further details reported.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Regression analysis computed with Statview (v.2)

Validity criteria fulfilled:

not applicable

Conclusions:

Growth of Tetrahymena pyriformis is inhibited to 50 % by 35, 20, and 20 mg Ti/L after 3, 6, and 9 h, respectively.

Executive summary:

The toxicity of Ti to Tetrahymena pyriformis was determined by using the flask technique. Samples with T. pyriformis in the exponential growth phase were incubated at least in triplicate for 9 hours with the test substance and growth inhibition of Tetrahymena pyrifoemis was determined by measuring the cellular density with a Coulter Counter.

Growth of Tetrahymena pyriformis is inhibited to 50 % by 35, 20, and 20 mg Ti/L after 3, 6, and 9 h, respectively.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 11

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Documentation insufficient for assessment. Justification for read-across: Due to similar or lower transformation/dissolution results for titanium carbide (the target substance) than titanium dioxide (the source substance), the resulting toxicity potential would also be expected to be similar or lower, so read-across is appropriate. Therefore, the dose descriptors are expected to be sufficiently similar or higher for the target substance, and read-across to the source chemical is adequately protective. For more details refer to the attached description of the read-across approach.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Vibrio fischeri NRRL B-11177 were exposed to TiO2 at 100 mg/L for up to 30 min in the Microtox test. To assess the effect of TiO2 on V. fischeri the light output of the sample was compared to the light output of the control. Measurements were made after 0, 5, 15, and 30 minutes.
In addition to the ecotoxicological experiments investigations regarding the actual particle size under test conditions were conducted (in Milli-Q water and natural pond water).

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Vibrio fisheri

Details on inoculum:

No details reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

30 min

Hardness:

No data.

Test temperature:

No data.

pH:

No data.

Dissolved oxygen:

No data.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concetrations: 0 (control) and 100 mg TiO2/L

Details on test conditions:

No details reported.

Reference substance (positive control):

no

Key result

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: fluorescence

Details on results:

- Toxic effects could not be observed, probably due to rapid aggregation and/or coagulation of TiO2 nanoparticles (aggregation/coagulation was confirmed by analytical measurements).
- Dilution experiments demonstrate that formation of larger particles and settling of the lager particles under normal gravitation or centrifugation occurred to a greater extent in natural (pond) water than in ultrapure (Milli-Q) water.
- Although fluorescence was measured up to 30 min. only results after 15 min. exposure are reported.

Validity criteria fulfilled:

not applicable

Conclusions:

Concentrations up to 100 mg TiO2/L did not effect the fluorescence of V. fischeri after 15 min of exposure.

Executive summary:

Vibrio fischeri were exposed to TiO2 nanoparticles at concentrations of 0 (control) and 100 mg/L for up to 30 minutes. Fluorescence as compared to the control was determined after 0, 5, 15, and 30 minutes.

In addition to the ecotoxicological experiments investigations regarding the actual particle size under test conditions were conducted (in Milli-Q water and natural pond water).

Under the conditions of the test fluorescence was not affected. The authors attribute the absence of ecotoxicologic effects to the rapid aggregation and/or coagulation of TiO2 nanoparticles resulting in (assumed) low bio-active concentrations of TiO2.

Reference 12

Endpoint:

toxicity to microorganisms, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to same study

Key result

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: fluorescence

Details on results:

- Toxic effects could not be observed, probably due to rapid aggregation and/or coagulation of TiO2 nanoparticles (aggregation/coagulation was confirmed by analytical measurements).
- Dilution experiments demonstrate that formation of larger particles and settling of the lager particles under normal gravitation or centrifugation occurred to a greater extent in natural (pond) water than in ultrapure (Milli-Q) water.
- Although fluorescence was measured up to 30 min. only results after 15 min. exposure are reported.

Validity criteria fulfilled:

not applicable

Conclusions:

Concentrations up to 100 mg TiO2/L did not effect the fluorescence of V. fischeri after 15 min of exposure.

Executive summary:

 

Vibrio fischeri were exposed to TiO2 nanoparticles at concentrations of 0 (control) and 100 mg/L for up to 30 minutes. Fluorescence as compared to the control was determined after 0, 5, 15, and 30 minutes.

In addition to the ecotoxicological experiments investigations regarding the actual particle size under test conditions were conducted (in Milli-Q water and natural pond water).

Under the conditions of the test fluorescence was not affected. The authors attribute the absence of ecotoxicologic effects to the rapid aggregation and/or coagulation of TiO2 nanoparticles resulting in (assumed) low bio-active concentrations of TiO2.

This information is used in a read-across approach in the assessment of the target sbstance. For justification of read-across please refer to the attachd read-across report (see IUCLID section 13).

Description of key information

The acute toxicity to aquatic microorganisms was tested using titanium dioxide and titanium tetrachloride. Hence, for titanium carbide this endpoint is derived by read-across from titanium dioxide and titanium tetrachloride.
The Tetrahymena growth inhibition test (Sauvant et al. 1995a, 1995b) shows the lowest effect value of EC50 (6 & 9 h) = 20 mg Ti/L.

Additional information

Sauvant et al (1995a, 1995b) examined the toxicity of TiCl₄ to Tetrahymena pyriformis by incubating the test organisms in the exponential growth phase for up to 9 hours with the test substance. Growth inhibition of Tetrahymena pyriformis was determined by measuring the cellular density. Growth of Tetrahymena pyriformis is inhibited by 50 % at 35, 20, and 20 mg Ti/L after 3, 6, and 9 h, respectively.

 

Adams et al. (2006) studied the toxic effects of TiO₂ (particle size 330 nm) to Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria E. coli with and without illumination (natural sunlight). The 14-h NOEC for TiO₂ (330 nm, illumination) in Bacillus subtilis was determined to be 500 ppm. The next higher concentration of 1000 ppm caused 75 % inhibition. The 14-h NOEC for TiO₂ (330 nm, illumination) in E. coli was determined to be 100 ppm. 44 ± 7.0 % and 46 ± 11.3 % inhibition could be observed at 1000 ppm and 2000 ppm, respectively. Illumination significantly (p < 0.05) enhanced toxicity of TiO₂ to Bacillus subtilis and E. coli, with growth inhibition being 2.5-fold and 1.8-fold greater in the presence of light, respectively. Thus, the authors conclude that the antibacterial effect of TiO₂ is related to the photocatalytic production of reactive oxygen species in addition to a supplementary mechanism as TiO₂ toxicity also occurs under dark conditions.

 

Inhibition of TiO₂ nanoparticles to Vibrio fischeri was investigated by measurement of luminescence (> Flash Assay) and growth (Heinlaan et al., 2008). Inhibition of the bacterial luminescence [%] was calculated after 30 min. NOEC (defined as < 20 % inhibition), EC20 and EC50 values were calculated. Subsequently, 72-h growth tests were conducted. Under the conditions of this study TiO₂ is not inhibitory to growth and luminescence of V. fischeri up to concentrations of 20,000 mg/L.

 

Effects of TiO₂ nanoparticles on Vibrio fischeri were also evaluated by Velzeboer et al. (2008) who exposed the test organisms at nominal concentrations of 0 (control) and 100 mg/L for up to 30 minutes. Fluorescence as compared to the control was determined after 0, 5, 15, and 30 minutes. Also in this test fluorescence was not affected.

 

According to ECHA Guidance R.7b "[...] single species tests with e.g. Vibrio fischeri (used in the MICROTOX® test), Pseudomonas fluorescens or Escherichia coli should be considered of low relevance for STPs. The tests with P. fluorescens and E. coli (Bringmann and Kühn 1960) cannot be used for determination of the PNECstp as they use glucose as a substrate (nor is E. coli a bacterium that will tend to multiply in an activated sludge environment). Likewise, Vibrio fischeri requires a high salinity environment. The information from such single-species screening tests may eventually be considered together with other existing data in a weight-of-evidence approach."

 

Available results were obtained in tests conducted above the solubility limit of the TiO₂. In general, according to ECHA Guidance R.7b "Microbial toxicity testing above the solubility limit of a chemical is to be avoided, similar to toxicity test with higher organisms. It is also unrealistic because insoluble chemicals will be removed in the primary settling tank or fat trap of full scale installations, and thus will not reach the activated sludge. However, data from existing tests where the experimentally derived NOEC is higher than the aqueous solubility can still be used as valid information to derive a PNECstp. This can be justified because it is a conservative estimate unlikely to occur in practice, and because undissolved test substance is found to be less confounding in microbial tests than in tests with higher organisms." Therefore, the available data are used for substance assessment.

 

Furthermore, ECHA Guidance R.7b states that "Ciliate-based test data can be used for deriving a PNECstp in case these are the sole data available or in multiple-data situations where the ciliates have the lowest NOEC." Thus, as the Tetrahymena growth inhibition test (Sauvant et al. 1995a, 1995b) shows the lowest effect value, the EC₅₀ (6 & 9 h) of 20 mg Ti/L is used in a WoE approach and is used as basis for substance assessment. 

 

Due to lower transformation/dissolution results for titanium carbide (the target substance) than titanium dioxide and titanium trichloride (the source substances), the resulting toxicity potential would also be expected to be lower. Therefore, the dose descriptors are expected to be sufficiently high for the target substance, and read-across to the source chemical is adequately protective. In fact, (eco-)toxicologically relevant release of Ti ions from titanium carbide is not expected as the concentration of soluble Ti ions was below the method detection limit (< 0.4 µg/L) in the T/D test. Thus, TiC is considered to be practically insoluble. Release of Ti ions to any ecotoxicologically relevant extent (and potential subsequent formation of soluble and/or insoluble Ti compounds) is not expected. Therefore, any toxic effects to aquatic microorganisms are not expected to arise from TiC and a PNECstp is not derived from the available data on TiCl₄.