Diiron titanium pentaoxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

For Read-Across Justification please refer to Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted: March 23, 2006

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material: Titanium dioxide (bulk)
- Molecular formula: TiO2
- Molecular weight: 79.87
- Supplier: Purchases in the testing facility (Sigma-Aldrich)
- Substance type: Solid
- Purity test date: 99.4%
- Lot No.: 70230
- Water solubility: Insoluble in water (<0.1 g/100 ml at 20℃)
- Storage condition of test material: Room temperature

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
0-hour sample at the start of the test: 15 mL taken from triplicate flask of each test concentration and pooled; 10 mL sample taken from pooled solution (3 replicates) and transferred into 20 mL volume vial; 0.3 mL of nitric acid solution (61%) added
72-hour sample at the end of the test: sample centrifuged (5,000 rpm, 10 min) to remove grown algae and prepared as described for 0-hour sample
- Sample storage conditions before analysis: no, samples were sent to the chemical analysis laboratory at Korea Research Institute of Chemical Technology for analysis of phosphorus

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: as the test substance is sparingly soluble in water, Water Accomodated Fraction (WAF) was prepared; 101 mg of the test substance was added in a 1000 mL beaker, OECD nutrient medium) added up to the 1000 mL mark of the beaker to get the nominal loading rate of 100 mg/L
- Stirring: stirred for about 40 minutes
- Filtering: filtered using a 0.45 µm membrane filter (Advantech MFS Inc., USA) to remove insoluble test substance
- Differential loading: a nominal limit loading rate of 100 mg/L was prepared
- Controls: negative control (OECD medium only)
- Evidence of undissolved material: no, a WAF was prepared and the test solution was filtered using a 0.45 µm membrane filter (see above)

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: UTEX 1648
- Source: Division of Non-clinical Studies, Korea Institute of Toxicology (KIT), Korea Research Institute of Chemical Technology (KRICT), Daejeon, Korea, originally purchased from UTEX (The Culture Collection of Algae, The University of Texas at Austin, USA) on August, 2006
- Pre-culture: sterile OECD nutrient medium (OECD, 2006) inoculated with about 5.0x10^3 cells/mL from stock culture two to four days before the test; to get algal growth in log phase the supsension was incubated under continuous illumination (light intensity: 60-120 µE/m^2/s) in a shaking incubator (shaking rate: about 100 times/min) at 21-24℃

ACCLIMATION
- Acclimation period: none

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

not reported

Test temperature:

23.4 +/-0.2 ℃

pH:

Control: 7.92 (0 hour) to 8.74 (72 hour)100 mg/L: 8.09 (0 hour) to 8.82 (72 hour)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal loading rate: 0 (negative control), 100 mg/L
Measured concentration: not detected, <0.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: not specified
- Material, size, headspace, fill volume: not specified
- Initial cells density: 5.0 x 10^3
- Control end cells density: 1.2 x 10^6 (mean)
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium acc. to guideline


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continiuous illumination
- Light intensity: 92 to 95 µE/m^2/s

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: electronic particle counter (BECKMAN COULTER Multisizer 3 particle analyzer)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable as a limit test was performed
- Range finding study: not specified

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: not reported
- Observation of abnormalities: no sign of contamination by foreign algal cells or protozoa
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no, a WAF was prepared and solutions were filtered previous to test

Results with reference substance (positive control):

Not specified

Reported statistics and error estimates:

Statistical analyses were not necessary since a limit test was performed.

Table 1. Cell densities ofPseudokirchnerella subcapitata (UTEX 1648) during the test period.

Nominal concentration (mg/L)	Mean measured concentration (mg/L)	Replicate No.	0-hour^1)	24-hour	48-hour	72-hour
Control	ND	R1	5.0 x 10^3	4.8 x 10^4	2.0 x 10^5	1.2 x 10^6
		R2	5.0 x 10^3	3.3 x 10^4	1.1 x 10^5	9.6 x 10^6
		R3	5.0 x 10^3	4.9 x 10^4	1.4 x 10^5	1.2 x 10^6
		R4	5.0 x 10^3	4.3 x 10^4	2.5 x 10^5	1.6 x 10^6
		R5	5.0 x 10^3	4.9 x 10^4	1.5 x 10^5	6.8 x 10^6
		R6	5.0 x 10^3	3.1 x 10^4	1.7 x 10^5	1.3 x 10^6
		mean	5.0 x 10^3	4.2 x 10^4	1.7 x 10^5	1.2 x 10^6
		SD	0	8.2 x 10^3	4.9 x 10^4	3.1 x 10^5
100	< LOQ	R1	5.0 x 10^3	3.8 x 10^4	1.9 x 10^5	1.4 x 10^6
		R2	5.0 x 10^3	2.4 x 10^4	1.6 x 10^5	1.3 x 10^6
		R3	5.0 x 10^3	4.1 x 10^4	2.3 x 10^5	1.5 x 10^6
		R4	5.0 x 10^3	4.7 x 10^4	2.5 x 10^5	1.8 x 10^6
		R5	5.0 x 10^3	3.5 x 10^4	2.0 x 10^5	1.5 x 10^6
		R6	5.0 x 10^3	3.5 x 10^4	1.4 x 10^5	1.4 x 10^6
		mean	5.0 x 10^3	3.7 x 10^4	2.0 x 10^5	1.5 x 10^6
		SD	0	7.7 x 10^3	4.1 x 10^4	1.7 x 10^5

1) Nominally inoculated value, ND: Not detected, LOQ(Limit of Quantitation): 0.1 mg/L

Table 2. Growth inhibition rates based on mean value during the test period; (1) average growth rate

Nominal concentration (mg/L)	Mean measured concentration (mg/L)	Average growth rate	Relative inhibition (%)	Mean relative inhibition (%)
Control	ND	1.8045^1)	-	-
100	< LOQ	1.8846	-4.4	-5.0
		1.8523	-2.7
		1.8981	-5.2
		1.9574	-8.5
		1.8902	-4.8
		1.8863	-4.5

1) Mean value of average growth rate in the control group, ND: Not detected, LOQ (Limit of Quantitation): 0.1 mg/L

Table 3. Growth inhibition rates based on mean value during the test period; (2) yield

Nominal concentration (mg/L)	Mean measured concentration(mg/L)	Yield	Relative Inhibition (%)	Mean relative inhibition (%)
Control	ND	1154000^1)	-	-
100	< LOQ	1422000	-23.2	-27.6
		1290000	-11.8
		1481000	-28.3
		1770000	-53.4
		1446000	-25.3
		1429000	-23.8

Table 4. pH

Nominal concentration (mg/L)	Mean measured concentration(mg/L)	0-hour	72-hour	Change of pH during the test period
Control	ND	7.92	8.74	0.82
100	< LOQ	8.09	8.82	0.73

ND: Not detected, LOQ (Limit of Quantitation): 0.1 mg/L

Table 5. Measured concentration of the test substance in the test solutions

Nominal concentration (mg/L)	Measured concentration (mg/L)		Mean measured cnocentraion (mg/L)
	0-hour	72-hour
Control	ND	ND	ND
100.0	< LOQ	< LOQ	-

Validity criteria fulfilled:

not specified

Conclusions:

In a Freshwater Alga and Cyanobacteria, Growth Inhibition Test according to OECD Guideline 201 (adopted: March 23, 2006) with Pseudokirchneriella subcapitata the 72-hr EL50 for for titanium dioxide (both growth rate and yield) were determined to be >= 100 mg/L in nominal.

Executive summary:

To assess the inhibitory effect of titanium dioxide on the growth of the unicellular green alga, Pseudokirchneriella subcapitata (Strain No. UTEX1648), a Freshwater Alga and Cyanobacteria, Growth Inhibition Test according to OECD Guideline 201 (adopted: March 23, 2006) was conducted in compliance with the GLP principles. As the test substance is sparingly soluble in water, a Water Accommodated Fraction according to OECD Guideline 23 (OECD, 2000) was prepared. The toxicity test was performed with a negative control (OECD medium only) and a nominal loading rate of 100 mg/L each in six replicates. The mean measured test item concentration was analyzed using ICP-MS to be below the Limit of Quantitation of 0.1 mg/L. Cultures were incubated on a shaking incubator under continuous illumination with a light intensity of 92 to 95 µE/m2/s at 23.4 +/-0.2 °C. The cell density of each replicate was measured by using a particle analyzer (BECKMAN COULTERTM Multisizer 3). Plots of growth curves were produced using the measured cell concentrations in test cultures and controls together with the concentrations of the test substance and the times of measurement. The inhibition percentage of the growth rate for each treatment was calculated based on both average growth rate and yield. In result, the 72 -hour EL50 for growth rate and yield was determined to be higher than 100 mg/L (nominal).

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

For Read-Across Justification please refer to Section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

15.6 other: mg Ti/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

35.9 other: mg Ti/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.1 other: mg Ti/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material: Titanium dioxide (Bulk) - Purchased: Sigma-Aldrich

Analytical monitoring:

not specified

Details on sampling:

not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: stock suspensions of the test item was prepared in algal medium by ultrasonication for 30 min immediately before the experiment
- Controls: not specified
- Evidence of undissolved material: not specified

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not specified

Hardness:

not reported

Test temperature:

24+/-1℃

pH:

not reported

Dissolved oxygen:

not reported

Salinity:

not applicable

Conductivity:

not reported

Nominal and measured concentrations:

not specified

Details on test conditions:

TEST SYSTEM
- Test vessel: incubation vials
- Material, size, fill volume: glass not, nominal volume: 20 mL, fill volume: 5 mL
- Initial cells density: 10000 cells/ mL
- Control end cells density: not specified
- No. of vessels per concentration: 3

GROWTH MEDIUM
- Standard medium used: not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not specified


OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: not specified
- Photoperiod: not specified
- Light intensity and quality: not specified

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: chlorophyll fluorescence; phase contrast as well as fluorescence micrographs were taken with Olympus GX41 microscope equipped with DP71 camera

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not specified
- Range finding study: not specified

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

15.6 other: mg Ti/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL = 12.0~19.3209

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

35.9 other: mg Ti/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL = 31.4~41.7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.1 other: mg Ti/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

not specified

Remarks on result:

other: 95% CL = 7.10~13.2

Reported statistics and error estimates:

The EC and NOEC with their confidence intervals were calculated using the Log-normal model of the REGTOX software for Microsoft Excel (Vindimian, 2005).

Table 1. The toxicity of bulk titanium dioxide to algae Pseudokirchnerella subcapitata (72hr growth inhibition).

Substance	EC50		EC20		NOEC
Bulk Titanium dioxide	mg Ti/L	95% Confidence interval	mg Ti/L	95% Confidence interval	mg Ti/L	95% Confidence interval
	35.9	31.4 ~41.7	15.6	12.0~19.3209	10.1	7.10~13.2

Validity criteria fulfilled:

not specified

Conclusions:

In an Alga, Growth Inhibition Test according to OECD Guideline 201 with Pseudokirchneriella subcapitata the 72-h EC50 (growth rate) and NOEC (basis not specified) for titanium dioxide was determined to be 35.9 mgTi/L and 10.1 mg Ti/L (measured, not specified).

Executive summary:

To assess the toxicity of titanium dioxide to Pseudokirchneriella subcapitata an Alga, Growth Inhibition Test according to OECD Guideline 201 was performed. In result the 72-h EC50 (growth rate) and NOEC (basis not specified) for titanium dioxide was determined to be 35.9 mg Ti/L and 10.1 mg Ti/L (measured, not specified).

Description of key information

The study on toxicity to aquatic algae and cyanobacteria with diiron titanium pentaoxide does not need to be conducted as the substance is highly insoluble in water and unlikely to cross biological membranes, hence indicating that aquatic toxicity is unlikely to occur. This conclusion is supported by assessments for the structural analogues diiron trioxide (CAS 1309-37-1) and titanium dioxide (CAS 13463-67-7) demonstrating that toxicity to aquatic algae is not to be expected. However, by applying an analogue approach the 72-hour EC50 and NOEC for the target substance were determined to be > 100 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Non submission justification for diiron titanium pentaoxide

No data on toxicity of diiron titanium pentaoxide to aquatic algae and cyanobacteria is available. The substance was assessed in three guideline studies to be sparingly soluble in water (references 4.8-2, 4.8-3 and 4.8-1). In a worst case approach the water solubility of the test item was determined to be 2.715 µg/L at a temperature of 20 °C (please refer to the endpoint summary in IUCLID section 4.8). Furthermore, the maximum of the mono-modal particle size distribution was observed at approximately between 10 µm and 20 µm. The D10, D50 and D90 percentile were determined as L10 = 7.07 µm, L50 = 13.1 µm and L90 = 22.9 µm, respectively. Considering these physicochemical properties, diirontitanium dioxide is unlikely to cross biological membranes (please also refer to IUCLID section 7.1). Therefore, in accordance with the criteria given in column 2 of Annex VII to Regulation (EC) No 1907/2006 (REACH), the study on toxicity to aquatic algae is considered scientifically not necessary and was waived.

Nevertheless, available data for the read across source substances titanium dioxide (CAS 13463-67-7) and diiron trioxide (CAS 1309-37-1) are summarized below.

In the following information on the toxicity of titanium dioxide (CAS 13463-67-7) and diiron trioxide (CAS 1309-37-1) to algae is provided.

Titanium dioxide

Data from two guideline studies with the source substance titanium dioxide (bulk form) is publicly available.

To assess the inhibitory effect of titanium dioxide on the growth of the unicellular green alga Pseudokirchneriella subcapitata (strain No. UTEX1648), a Freshwater Alga and Cyanobacteria, Growth Inhibition Test according to OECD Guideline 201 (adopted: March 23, 2006) was conducted in compliance with the GLP principles. As the test substance is sparingly soluble in water, a Water Accommodated Fraction according to OECD Guideline 23 (OECD, 2000) was prepared. The toxicity test was performed with a negative control (OECD medium only) and a nominal loading rate of 100 mg/L each in six replicates. The mean measured test item concentration was analyzed using ICP-MS to be below the Limit of Quantitation of 0.1 mg/L. Cultures were incubated on a shaking incubator under continuous illumination with a light intensity of 92 to 95 µE/m2/s at 23.4 +/-0.2 °C. The cell density of each replicate was measured by using a particle analyzer (BECKMAN COULTER^TM Multisizer 3). Plot of growth curves were produced using the measured cell concentrations in test cultures and controls together with the concentrations of the test substance and the times of measurement. The inhibition percentage of the growth rate for each treatment was calculated based on both average growth rate and yield. In result, the 72-hour EL50 for growth rate and yield was determined to be higher than 100 mg/L in nominal (reference 6.1.5 -1).

In another Alga, Growth Inhibition Test according to OECD Guideline 201 with Pseudokirchneriella subcapitata the 72-hour EC50 (growth rate) and NOEC (basis not specified) for titanium dioxide was determined to be 35.9 mg Ti/L and 10.1 mg Ti/L (measured, not specified) (reference 6.1.5 -2). This information is considered not reliable since the documentation is insufficient for assessment.

Key information on short-term toxicity to fish provided in public ECHA REACH dossiers for the read across substances titanium dioxide and diiron trioxide

Titanium dioxide

In the public ECHA REACH dossier on titanium dioxide (last accessed on: 18 March 2020) dispersions of microsized TiO2 were found to be not toxic to aquatic algae and cyanobacteria at a concentration of at least ≥ 100 mg/L (NOEC) up to 5600 mg TiO2/L (NOEC) in fresh- and saltwater, respectively. Based on this results and taking into account microsized TiO2 is sparingly soluble in water, microsized titanium dioxide is considered to be not toxic to freshwater algae up to its solubility limit. A low toxic potential of algal toxicity is observed for dispersed nanosized titanium dioxide with EC10 and EC50 values being > 2 mg/L and > 50 mg/L, respectively. However, the read across hypothesis for the registration substance diiron titanium pentaoxide (CAS 12023-27-7) only comprises titanium dioxide in its bulk form, but no nanoparticles.

Based on the available information and the public assessment in the ECHA REACH dossier it is concluded that the source substance titanium dioxide has no adverse effect to algae and cyanobacteria.

Diiron trioxide

In the public ECHA REACH dossier on diiron trioxide (last accessed on: 18 March 2020) the data requirement on toxicity to algae was waived in accordance with column 2 of REACH Annex VII of Regulation (EC) 1907/2006 (REACH) since the addressed category members are all inert inorganic oxides of iron, manganese and zinc and highly insoluble in water. Moreover, it is stated that soluble salts of respective metals are essential nutritions of algal growth and proposed test media contain these compounds at concentrations exceeding the solubility of iron in the iron oxides. Consequently, diiron trioxide is not expected to be toxic to algae and cyanobacteria.

Conclusion

Based on the physico-chemical properties of diiron titanium pentaoxide a test on toxicity to algae or cyanobacteria is scientifically not justified in compliance with column 2 of Annex VII to Regulation (EC) No 1907/2006 (REACH). Moreover, the source substances titanium dioxide (CAS 13463-67-7) and diiron trioxide (CAS 1309-37-1) are found not to be toxic to aquatic algae. Both structural analogues are neither classified for short-term (acute) nor long-term (chronic) aquatic toxicity according to Regulation (EC) No 1272/2008 (CLP).

Target and source substance are metal-containing inorganic oxides. The target substance diiron titanium pentaoxide is a mixed oxide of titanium dioxide and diiron trioxide. The source substances share the common atoms Ti, O and Fe with the target substance and have very similar physico-chemical properties (for further information please refer to the read across justification attached to IUCLID section 13). Therefore, by applying an analogue approach, diiron titanium pentaoxide is also considered to be not toxic to aquatic algae and cyanobacteria and further studies are thus scientifically not required. Following a weight of evidence approach the 72-hour EC50 and NOEC (growth rate) for the target substance are determined to be > 100 mg/L.