Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other:
Remarks:
TiO2 concentrations measured in exposure medium; Applied spacing factor of 10 and testing of only two concentrations; additional tests performed under food depleting conditions and/or performed with a too short exposure period of only 9 or 11 d; organic extract in test medium not described; light intensity not specified
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 202
Version / remarks:
1981 / 21 day reproduction test (including acute immobilization tests)
Principles of method if other than guideline:
Experiment 1a was performed according to OECD 202 (1981). Furthermore, additional experiments were performed which were not conducted according to OECD 202 (2012). These tests were performed under food depleting conditions (experiment 1b, 2b and 3b), or with a shorter test duration of only 9-11 d (experiment 2a/b and 3a/b). Hence, results of these studies are not considered for the hazard assessment of nano-TiO2. A spacing factor of 10 was used and only two concentrations were tested.
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
yes
Details on sampling:
Particles were dispersed in ASTM hard synthetic water (pH 7.8) following the described dispersion method. Thereafter, suspensions were left to settle for 24 h and water samples were taken.
Vehicle:
no
Details on test solutions:
- Direct addition of the test material into test vessel (dispersion): no
- Sonication of stock dispersion: Initial stock dispersions of particle suspensions of 1 g/L were dispersed in a ultrasonic bath during 30 min in MilliQ water and left for 24 h in agitation in darkness prior to be used to prepare test solutions (1 and 10 mg/L).
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: dispersions in ASTM medium were renewed every 24 h
- Test medium: ASTM hard synthetic water
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: clone F
- Feeding during test: yes
- Food type: C. vulgaris
- Amount:
150 µg C per daphnid per day (experiment 1a; experiment 2a and 3a)
30 µg C per daphnid per day (experiment 1b; experiment 2b and 3b)
- Frequency: daily
- Photoperiod: 14 h light: 10 h dark
- Temperature: 20 ± 1 °C
- Light source: neon fluorescent lamp (UVA: < 10 µW/cm²)

ACCLIMATION
- Acclimation period: culture was maintained in ASTM hard synthetic water
- Type and amount of food: same as in test
- Feeding frequency: same as in test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
no information
Test temperature:
20 ± 1 °C
pH:
7.8
Dissolved oxygen:
no information
Salinity:
--
Conductivity:
--
Nominal and measured concentrations:
Nominal (NM 103, NM 104, P25): 1 mg/L and 10 mg/L
Measured 24 h (NM 103): 0.81 ± 0.08 mg/L and 2.80 ± 0.30 mg/L
Measured 24 h (NM 104): 0.93 ± 0.1 mg/L and 3.12 ± 0.21 mg/L
Measured 24 h (P25): 0.8 ± 0.06 mg/L and 2.70 ± 0.28 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
Experiment 1: 120 ml screw top glass jars (6 cm water column)
Experiment 2 and 3: 1.2 L screw top glass vessels (16 cm water column)
- Aeration: no aeration
- Renewal rate of test solution (frequency): every 24 h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ASTM hard synthetic water with organic extract (not specified)
- Culture medium different from test medium: no, both ASTM hard synthetic water

OTHER TEST CONDITIONS
- pH: 7.8
- Photoperiod: 14 h light: 10 h dark
- Light intensity: UVA < 10 µW/cm²
- Light source: neon fluorescent lamps

EFFECT PARAMETERS MEASURED:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

TEST SET UP
- Experiment 1:
Food conditions: non-depleting (a) and depleting (b)
water column height: 6 cm
Shaking: no
Test duration: 21 d

- Experiment 2:
Food conditions: non-depleting (a) and depleting (b)
water column height: 16 cm
Shaking: no
Test duration: 9-11 d

- Experiment 3:
Food conditions: non-depleting (a) and depleting (b)
water column height: 16 cm
Shaking: yes, 2 rpm
Test duration: 9-11 d
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
for NM 103, NM 104 and P25
Basis for effect:
other: Age at first brood; cummulative fecundity; body length at 21 d, population growth rate, size of first brood
Remarks on result:
other: Experiment 1a
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 2.8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: dispersed in water phase
Remarks:
measured after 24 h (n=4); for NM 103
Basis for effect:
other: Age at first brood; cumulative fecundity; body length at 21 d, population growth rate, size of first brood
Remarks on result:
other: Experiment 1a
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 3.12 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: dispersed in water phase
Remarks:
measured after 24 h (n=4); for NM 104
Basis for effect:
other: Age at first brood; cummulative fecundity; body length at 21 d, population growth rate; size of first brood
Remarks on result:
other: Experiment 1a
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 2.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: dispersed in water phase / measured after 24 h
Remarks:
for P25
Basis for effect:
other: Age at first brood; cummulative fecundity; body length at 21 d, population growth rate, size of first brood
Remarks on result:
other: Experiment 1a
Details on results:
Experiment 1 a:
As supplementary endpoint, body length of adults at first maturity was investigated. It was shown that this endpoint was significantly reduced compared to control organisms, when organisms were exposed to 10 mg P25/L (nominal).
However, body length of adults after 21 d was not significantly affected at 10 mg P25/L (nominal) compared to control organisms. This endpoint is recommended in OECD 211 to assess effects on growth of D. magna. Furthermore, the present study shows that the observed effects of P25 on body length of adults at first maturity have no influence on the age at first brood, survival of adult organisms, and the cumulative fecundity, or on fitness measurements such as population growth rate. These endpoints are the most sensitive and important endpoints which have to be considered according to OECD 211 and ECHA Guidance, Chapter R. 7b.
Body length of adult organisms at first maturity was not affected when organisms were exposed to all other test materials up to the highest test concentration (10 mg/L, nominal).

Experiment 1b (tested under food depleting conditions; not reliable):
- P25 significantly lowered the size of first brood at 10 mg/l compared to control, but not at 1 mg/L.
- NM 103 significantly reduced size of first brood, population growth rate and significantly enhanced age at first brood, as well as body length of adults at first maturity at 10 mg/L compared to control.

Experiment 2a (1 L test vessels + non-depleting food conditions; test duration 9-11 d; not reliable):
- Only P25 significantly reduced size of brood, body length of adults and population growth rate and significantly enhanced age at first brood when daphnids were exposed to 10 mg/L compared to control.

Experiment 2b (1 L test vessels + depleting food conditions; test duration 9-11 d; not reliable):
- All materials significantly reduced brood size and population growth rate at 1 and 10 mg/L compared to control.

Experiment 3 a (1 L test vessels, non-depleting food conditions, resuspension, test duration 9-11 d; not reliable):
- P25 significantly reduced size of brood and population growth and significantly enhanced age at first brood at 10 mg/L compared to control.
- NM 103 significantly reduced population growth rate and significantly enhanced age at first brood at 10 mg/L compared to control.

Experiment 3b: (depleting food conditions; 1 L test vessels, resuspension, test duration 9-11 d; not reliable):
- All test materials significantly reduced brood size and population growth rate at 10 mg/L compared to control.
Reported statistics and error estimates:
Each experiment and food ration was analyzed separately by one way ANOVA to exclude confounding effects of inter-trail/experiment variation. When significant (P<0.05) treatment effects occurred, post hoc Tukey's or Dunnet's tests were performed to compare treatments. Prior to analyses all data except age at first reproduction were log transformed to improve normality and variance homoscedasticity. Effects of treatment on age at first reproduction were analyzed by non-parametric Kruskal-Wallis test. Analyses of r were based on jackknife pseudovalues. Tests were conducted with the aid of the statistical package IBM SPSS v19.
Validity criteria fulfilled:
yes
Conclusions:
Dispersions (1 and 10 mg/L, nominal) of the nano-sized TiO2 materials NM 103 (20 nm, rutile, hydrophobic coating), NM 104 (20 nm, rutile, glycerin coating), and P25 (21 nm, rutile-anatase) did not affect the age at first brood, size of first brood, cumulative fecundity, body length of adult females after 21 d, or the population growth rate of Daphnia magna in semi-static, chronic toxicity tests according to OECD 202 (1981). Derived 21 d-NOEC values for these endpoints amounted to ≥ 10 mg/L (nominal) for all materials and ≥ 2.80, 3.12, and 2.70 mg/L when based on measured concentrations of NM 103, NM 104 and P25.
Only high levels of P25 (10 mg/L, nominal) significantly reduced body length of adult organisms at first maturity. However, body length of adults after 21 d was not significantly affected at 10 mg P25/L (nominal) compared to control organisms. This endpoint is recommended in OECD 211 to assess effects on growth of D. magna. Furthermore, the present study shows that the observed effects of P25 on body length of adults at first maturity have no influence on the age at first brood, survival of adult organisms, and the cumulative fecundity, or on fitness measurements such as population growth rate. These endpoints are the most sensitive and important endpoints which have to be considered according to OECD 211 and ECHA Guidance, Chapter R. 7b.
Body length of adult organisms at first maturity was not affected when organisms were exposed to all other test materials up to the highest test concentration (10 mg/L, nominal).

Results of additional experiments are not relevant for the hazard assessment of nano-TiO2, because they were either performed under depleting food conditions (experiments 1b, 2b and 3b), which are expected to enhance daphnia sensitivity, or were conducted with a shorter test period than that recommended in OECD 202 (1981, experiment 2a/b and 3a/b).
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test concentration of the sediment and pore water was not analysed. Overlaying water was spiked and analysed.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 219 (Sediment-Water Chironomid Toxicity Test Using Spiked Water)
Deviations:
no
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:
yes
Details on sampling:
For the control and for each concentration one additional vessel was used for analytical measurements. The additional vessels were treated as the control vessels and the test vessels used for the assessment of the nanoparticles.
At 1, 7, 14 and 28 d of exposure aqueous samples (50 mL) were taken at four depths (about 2.0 cm; 4.0 cm; 5.5 cm; 6.5 cm). The samples were combined. About 20 mL was used for analysis and the remaining amount was carefully returned into the test vessels without disturbing the sediment.
Vehicle:
no
Details on test solutions:
- Direct addition of the test material into test vessel: no
- Sonication of dispersion: For each vessel, a 500 mL double concentrated stock dispersion of the nanomaterial was prepared in tap water. For the double concentrated dispersion of the final test concentration the respective amount of nanomaterial was weighed in brown glass vessels using a suitable
balance. Five hundred millilitres of tap water was added, the mixture was stirred (magnetic stirrer, 900 rpm), followed by ultrasonic treatment in a water bath (3 min, 500 W). The double concentrated stock dispersion was added thoroughly to the water column (500 mL) in the test vessels 24 h after adding the test specimens.
- Appearance of dispersion: Due to the activity of the chironomids in the sediment, settled TiO2 nanoparticles were transferred into the sediment. During the exposure period of 28 days the settled white powder of TiO2 on the surface of the sediment vanished.
- Renewal of test dispersion: no, static
- Test medium: Purified tap water
Test organisms (species):
other: Chironomus riparius
Details on test organisms:
TEST ORGANISM
- Source: Bayer Crop Science AG (Monheim, Germany)
- Breeding conditions:Purified tap water was added to a layer of diatomaceous earth.
- Feeding during test
- Food type: powder of TetraMin® Hauptfutter (Tetra Werke, Melle, Germany)
- Frequency: the dipterans were fed daily

ACCLIMATION
- Acclimation period: 5 days before treatment
- Acclimation conditions: the egg masses were taken from the cultures and placed in small aerated vessels with test water at about 20 °C.
Test type:
static
Water media type:
freshwater
Remarks:
and artificial sediment
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
No post-exposure observation period was performed.
Hardness:
P25:
Test start: 110 – 150 mg/L as CaCO3 equivalents
Test end: 210 mg/L as CaCO3 equivalents in one representative replicate (100 – 120 mg/L as CaCO3 equivalents in the controls)
NM-101:
Test start: 140 mg/L CaCO3 equivalents in one representative replicate (130 – 150 mg/L CaCO3 equivalents in the control)
Test end: 170 mg/L CaCO3 equivalents in one representative replicate (150 – 170 mg/L CaCO3 equivalents in the control)
Test temperature:
20 ± 2°C
pH:
permitted range: pH 6 – 9
Dissolved oxygen:
About 100% at test start and test end (demanded threshold value: 60%)
Salinity:
not required
Nominal and measured concentrations:
nominal concentrations: 15, 23, 39, 63, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test container (material, size): round glass beaker
- Sediment volume: 3 L
- Weight of sediment: 370 g dry mater
- Depth of sediment and overlying water: 2 cm sediment and 8 cm overlaying water
- Aeration: yes
- Aeration frequency and intensity: gentle

EXPOSURE REGIME
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- Type and preparation of food: TetraMin® Hauptfutter powder
- Amount of food: 0.25 – 0.5 mg TetraMin® /larvae/day (day0-10); from day 10 on the food ration was increased to 0.5 – 1.0 mg TetraMin® /larvae/day.

OVERLYING WATER CHARACTERISTCS
- Type of water: purified tap water

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 5%
- Composition: Artificial sediment components: Kaolinite, air-dried 20%; Industrial quartz sand, air-dried 75%
- Moisture: water content 25 - 30 %
- Total organic carbon (%): 2 % ± 0.5 %

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark
- Light intensity: 500 - 1000 lux
Reference substance (positive control):
yes
Remarks:
2-chloracetamid
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25; Hombikat UV 100
Basis for effect:
other: emergence rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25; Hombikat UV 100
Basis for effect:
other: development rate
Reported statistics and error estimates:
The number of emerged males and females were determined. The results of the listed biological parameters (total, males, females) were compared by a suitable test for multiple comparisons with a control after testing variance homogeneity. All statistical tests were performed with the computer software ToxRat Professional version 2.10.4.1 (ToxRat® Solutions GmbH).

TITANIUM CONCENTRATIONS

P25 - Ti concentrations in overlaying water:

- Measured Ti concentrations after 1 d of expsoure (nominal: 0, 9.0, 14.4, 23.4, 37.8, 59.9 mg Ti/L): 11.8, 518.7, 627.2, 651.2, 466.4, 471.2 µg Ti/L

- Measured Ti concentrations after 28 d of expsoure in treatment groups (nominal: 0, 9.0, 14.4, 23.4, 37.8, 59.9 mg Ti/L): all Ti concentrations decreased below the limit of detection (no value given)

Hombikat UV 100 - Ti concentrations in overlaying water:

- Measured Ti concentrations after 1 d of expsoure (nominal: 0, 9.0, 14.4, 23.4, 37.8, 59.9 mg Ti/L): 6.7, 825, 1163, 1626, 1639, 1312 µg Ti/L

- Measured Ti concentrations after 28 d of expsoure in treatment groups (nominal: 0, 9.0, 14.4, 23.4, 37.8, 59.9 mg Ti/L): all Ti concentrations decreased to or were lower than the Ti background level in controls (3.17 µg Ti/L)

Hombikat UV 100 - sediment concentrations:

- due to the high background Ti concentrations in the sediment it was not possible to determine valid measurments in sediment samples

DLS RESULTS (Z-average)

- P25 in test medium at test start and after 1 d of exposure: 970 nm-2262 nm

- Hombikat UV 100: no measurements performed

Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the nano sized TiO2 materials P25 (anatase, rutile, 21 nm) and Homibkat UV 100 (anatase, 8 nm), which were applied via the overlaying water to the test system were tested in a chronic sediment toxicity test according to OECD 219. Both materials did not affect the emergence and development rate of Chironomus riparius, resulting in 28 d-NOEC values of ≥ 100 mg n-TiO2/L (nominal). For Homibkat UV 100 all validity criteria were fulfilled. The mean emergence in the controls of the P25 test was 68% at test end. The miniumum as stated in the guideline is 70%. As deviation is only minor and all other validity criteria are fullfilled and P25 induced any effects, this test was not repeated.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: following the principles of GLP (quality assurance unit not involved, no archiving of data; laboratory equipment was controlled and documented according to GLP)
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
Three experiments were performed:
Experiment 1: medium renewal three times a week
Experiment 2: medium renewal three times a week and daily
Experiment 3: medium renewal three times a week, sonication period 3 and 30 min
In the first test the results differed from those reported in the literature. Experiment 2 and 3 were performed for clarification of the discrepancies.
GLP compliance:
no
Remarks:
Following the principles of GLP (quality assurance unit not involved, no archiving of data; laboratory equipment was controlled and documented according to GLP
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Samples for analyses were taken from the stock dispersion of each concentration before test start, and from the test vessels after the incubation periods listed below, and at each medium renewal. Samples from the incubated test suspensions were withdrawn from the water phase. Care was taken that sedimented particles were not removed. Sampling times were as follows:

Medium renewal three times a week
• Day 0 (start of the test), 7, and 14: stock dispersion and each freshly prepared test dispersion
• Day 2, 9, and 16: each test concentration after incubation (day 2: before medium renewal) in the test vessels with daphnids
Medium renewal daily
• Day 0 (start of the test), 7, and 14: stock dispersion and each freshly prepared test dispersion
• Day 1,8, and 15: each test concentration after incubation (day1.:before medium renewal) in the test vessels with daphnids

- Sample method for ICP-MS: Prior to digestion, the aqueous sample containing TiO2 was vigorously shaken (e. g. by a vortexer) for at least 1 min. Directly afterwards 4 mL were taken and 1 mL of a mixture of hydrochloric-, nitric-, and hydrofluoric acid in a ratio of 3:1:1 was carefully added.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Direct addition of test material into test dispersion: no
- Sonication of dispersions: Purified tap water was used as test water and to prepare the test suspensions. Stock dispersion (20 mg/L) and every test dispersion was stirred (magnetic stirrer, 900 rpm) and treated with ultrasound in a water bath (Experiment 1 and 2: 3 min, 500 W; Experiment 3: additional 30 min, 500 W). Stock dispersions were freshly prepared before each renewal period.

The test concentrations were achieved by dilution:
5.0 mg/L: 250 mg/L stock dispersion + 750 mL purified tap water
1.0 mg/L: 50 mg/L stock dispersion + 950 mL purified tap water
0.5 mg/L: 25 mg/L stock dispersion + 975 mL purified tap water
0.1 mg/L: 5 mg/L stock dispersion + 995 mL purified tap water
0.05 mg/L: 2.5 mg/L stock dispersion + 997.5 mL purified tap water

- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersions:
Experiment 1: medium renewal three times a week, sonication period 3 min
Experiment 2: medium renewal three times a week and medium renewal daily, sonication period 3 min
Experiment 3: medium renewal three times a week, sonication period 3 and 30 min
- Test medium: purified tap water
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene; Specimens used in the test were bred in the laboratory of the Fraunhofer IME.
- Age of parental stock (mean): at least 3 weeks-old
- Feeding during test
- Food type: algal suspension (Desmodesmus subspicatus) and Liquizell (HOBBY)
- Amount: 30 mL of algae pellet (Algae growing in log-phase) to 1L medium (The content of food in the test suspensions, measured as turbidity at 758 nm, was increased from about 7 mg C/L equivalents to 15 mg C/L equivalents.)
- Frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
New born Daphnia were separated by sieving, the first generation was discarded.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
total: 1.1 mmol/L
Test temperature:
20 ± 2°C
pH:
7.8 – 8.8
Dissolved oxygen:
About 100% corresponding to about 8.6 mg/L (demanded threshold value: 3 mg/L)
Salinity:
--
Conductivity:
304 - 337 µS/cm
Nominal and measured concentrations:
nominal concentrations (1st experiment): 0, 0.05, 0.1, 0.5, 1.0, 5.0 mg TiO2/L
nominal concentrations (2nd & 3rd experiment): 0, 1.0, 5.0 mg TiO2/L

1st experiment:
Recovery in stock dispersion (20 mg/L): 87.2%-89.8% of the nominal concenration
Recovery in freshly prepared test dispersions (nominal:0.05-5 mg/L): 64 - 86% of the nominal concentrations
Recovery in test dispersions after two days of incubation (nominal: 0.05-5 mg/L): 2.8-18.0% after two days of incubation

2nd experiment:
Recovery in stock dispersion (20 mg/L): 84.6-101.3% of the nominal concenration
Recovery in freshly prepared test dispersions (nominal: 1-5 mg/L): 22.6-49.3% of the nominal concentrations
Recovery in test dispersions after two days of incubation (nominal: 1-5 mg/L): 8.6-38.4% after two days of incubation

3rd experiment:
The applied test concentrations were not validated. The test suspensions were prepared following the procedure applied for the first two tests, except that for one experiment the sonication period was 30 min compared to 3 min.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers (60 mL) filled with 50 mL test suspension
- Type (delete if not applicable): closed (covered with glass panes)
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 1st test: 3 times a week; 2nd test: three times a week and daily; 3rd test: three times a week, sonication period 3 and 30 min
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

1st EXPERIMENT MEDIUM / WATER PARAMETERS
- Total organic carbon: 0.9 mg/L
- Metals: Cd, Pb, Ni, Mn < LOQ; Zn 4.7-5.8 µg/L; Fe - Chlorine: <0.02 mg/L
- Alkalinity: 2.1-2.9 mmol/L
- Ca/mmol ratio: 0.9

- Conductivity: 304 - 337 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: once per month

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: : light/dark cycle 16/8 h
- Light intensity: 563 - 591 lux

2nd EXPERIMENT MEDIUM / WATER PARAMETERS (diffent parameters)
- Total organic carbon: 0.9 mg/L
- Metals (µg/L): Cd < LOQ; Cr <1.96; Cu <6.24; Fe <8.57; Mn <2.26; Ni <1.26; Pb <9.50; Zn < 15.2
- Chlorine: <0.02 mg/L
- Alkalinity: 2.1 mmol/L
- Ca/mmol ratio: 0.8
- Conductivity: 298 µS/cm
- Light intensity: 560 - 607 lux

3rd EXPERIMENT MEDIUM / WATER PARAMETERS (diffent parameters)
- Total organic carbon: mg/L
- Metals: Cr <1.96; Cu <6.24; Fe <8.57; Mn <2.26; Ni <1.26; Pb <9.50; Zn 5.16 – 10.5
- Chlorine:<0.02 – 0.02 mg/L
- Alkalinity: 2.1 – 2.4 mmol/L
- Ca/mmol ratio: 0.9 – 1.0

- Conductivity: 314- 326 µS/cm
- Light intensity: 811 - 891 lux

Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
reproduction
Remarks on result:
other: NOECs derived in three of five independent tests
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other:
Remarks:
LOECs derived in two of five independent experiments
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
other: immobilisation/ body length
Remarks on result:
other:
Remarks:
NOECs derived in five of five independent tests
Details on results:
Experiment 1:
LOEC, EC10, EC20, and EC50 values of the biological endpoints (cumulative offspring per survivor, mobility) were > 5 mg/L. NOEC was ≥ 5 mg/L. Although body length was significantly lower in treatments with TiO2 concentrations higher and equal to 0.5 mg/L, the maximum effect was only 10% and no concentration effect relationship was observed. Therefore, it is concluded that P25 up to a concentration of 5 mg/L does not affect body length and the statistical difference is based on the very homogeneous length and the high number of replicates.

Experiment 2:
LOEC, EC10, EC20, and EC50 values of the biological endpoints (cumulative offspring per survivor, mobility, and body length) were > 5 mg/L. NOEC was ≥ 5 mg/L.

Experiment 3:
For mobility and body length, NOEC values were ≥ 5.0 mg/L, and LOEC values were > 5.0 mg/L. The mean cumulative offspring per female was significantly reduced by 10% and 16% at 5 mg/L when the TiO2 dispersion was ultrasonicated for 3 min and 30 min, respectively. Thus, the LOEC was 5.0 mg/L for both tests.
A NOEC should not be derived from these two experiments since the separation factor should not exceed 3.2 according to OECD 211. No significant difference between ultrasonication periods of 3 min and 30 min, as well as between 1 and 5 mg/L treatment groups was observed.

In experiment 3, no other clinical signs were detected in any replicate at any concentration tested. The calculated differences for the cumulative offspring per female should not be overestimated due to the fact that: (i) only two concentrations were tested and the reliability check via concentration-effect relationships was not possible; (ii) the results of 3 min and 30 min ultrasonication did not significantly differ; and (iii) only in two out of five tests, although significant, minor effects on reproduction (10-16%) were observed at 5 mg/L.
Reported statistics and error estimates:
Statistical method
In this report numerical values are frequently rounded to a smaller degree of precision (number of digits) than have been used in the actual calculation. Minor differences in results obtained from calculations with rounded values compared to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and of no practical concern. The parental mortality, the time to first brood and the number of offspring were used to calculate the intrinsic rate of population increase r as integrative parameter relevant for population effects.

Statistical calculations
The results of the listed biological parameters were compared by a suitable test for multiple comparisons with a control after testing variance homogeneity. All statistical tests were performed with the computer software ToxRat version 2.10.4.1 Professional (ToxRat® Solutions GmbH).
Validity criteria fulfilled:
yes
Remarks:
survival in the control (100%) was above 80%; in 21d the mean number of offspring in the control was above the criterion of 60/female
Conclusions:
Daphnids (aged < 24 h at test start) were exposed to nano sized TiO2 dispersions of P25 (21 nm) in five independent semi-static chronic toxicity tests according to OECD 211 guideline in which the media was either renewed daily or three times per week. 21 d-NOEC values ≥ 5 mg/L (nominal, highest concentration testedl) and 21 d-LOEC values > 5 mg/L (nominal) were determined for mortality, mobility and body length in all experiments. In three out of five experiments 21 d-NOEC and LOEC values for reproduction were ≥ 5 and > 5 mg/L, respectively. Only in two out of five experiments reproduction was significantly reduced by 10-16% at 5 mg/L, resulting in a LOEC of 5 mg/L. A NOEC should not be derived from the latter two experiments since the separation factor should not exceed 3.2 according to OECD 211. It is recommended not to overestimate the results of the latter tests and to consider the whole available data of all five tests.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Sediment and pore water were not analysed; overlaying water was spiked and analysed.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 225 (Sediment-Water Lumriculus Toxicity Test Using Spiked Sediment)
Deviations:
yes
Remarks:
water phase instead of sediment was spiked
Principles of method if other than guideline:
The test substance was applied to the overlaying water and was not spiked directly to the sediment as recommended in the guideline. Two experiments were performed, in the first experiment concentrations between 15-100 mg TiO2/L were tested and in the second experiment only 100 mg TiO2/L was tested. Only in the second experiment TiO2 concentration in the overlaying water were analysed.
GLP compliance:
no
Remarks:
But the test followed GLP principles (laboratory equipment was controlled and protocolled according to GLP, no quality assurance unit involved).
Analytical monitoring:
yes
Details on sampling:
SEDIMENT
Sediment was not included in analytical monitoring.

OVERLAYING WATER
- Sampling interval: 1, 7, and 28 d after exposure
- Sample storage before analysis: refrigerator
- Sampling: In each replicate three sub samples of 5 ml were taken and pooled. One sub sample was taken from approximately one third and one from two third of the height of the test medium, the third sample was taken from 1 cm above the sediment.
- Number of replicates: maximum two replicates per control/treatment group
Vehicle:
no
Details on test solutions:
- The test vessels were filled with sediment including the test organisms and 50% of the volume of the overlaying water one day before test start. On the following day, dispersions of the test substance were prepared and adminstered into the test vessles to achive the nominal concentrations. Immediately after application, test media were stirred with a glass rod.

PREPARATION OF STOCK DISPERSIONS:
- Direct addition of test material into test vessel: no
- Sonication of stock dispersion: The desired test amount was weighed into a glass vessel containing the appropriate volume of dionized water. Dispersions were stirred for 60 seconds on a magnetic stirrer (900 rpm) and placed into an ultrasonic water bath filled to one third of the dispersion height in the bottles (Badelin Sonorex RK 514 BH; 35 kHz; 215/860 W) for three minutes.
- Solvent: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of test dispersion: no information
- Renewal of test dispersion: no, static
- Test medium: Reconstituted freshwater (OECD 203)
Test organisms (species):
other: Lumbriculus variegatus
Details on test organisms:
TEST ORGANISM
- Common name: blackworm
- Source: Fischfutter Etzbach (Mechernich-Bergheim, Germany)
- Food type: the worms were fed with fish food suspension (50 g/L TetraMin ® )
- Frequency:daily
Test type:
static
Water media type:
freshwater
Remarks:
and artificial sediment
Limit test:
yes
Total exposure duration:
28 d
Hardness:
178 – 267 mg/L CaCO 3
Test temperature:
20 ± 2°C
pH:
7.5 – 8.0
Nominal and measured concentrations:
nominal concentrations (sediment overlying water): 15; 23; 39; 63 and 100 mg NM-105/L
Details on test conditions:
TEST SYSTEM
- Test container (material, size): glas vessels, 250 mL total volume with plastic lid
- Weight of wet sediment: 80 g wet weight
- Overlying water volume: 180 mL
- Depth of sediment and overlying water: ca. 1.5 cm height of sediment in test vessel
- Aeration: yes
- Aeration frequency and intensity: continuous aeration
- Replacement of evaporated test water, if any: static; 3 days per week adjustment for evaporated test medium

EXPOSURE REGIME
- No. of organisms per container (treatment): 10 worms
- No. of replicates per treatment group: 4
- No. of replicates per control: 6
- Feeding regime: food in sediment
- Type and preparation of food: Alpha-cellulose and utrica powder) was mixed into the sediment

OVERLYING WATER CHARACTERISTCS
- Type of water: reconstituted water are according to OECD TG No. 203
- Conductivity: 550 – 650 µS/cm
- Oxygen saturation: > 80%

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 5 ± 0.5
- Particle size distribution: 402-1325 nm (declining over time)

- Composition (if artificial substrate):
- % sand: 75-76
- % clay: 20 ± 1
- Method of preparation (if artificial substrate):
- Maturation of artificial substrate (if any): yes/no
- pH dry matter and/or whole sediment: 6.4
- Ammonia content of pore water: <0.01 mg/L
- Total carbon (%): 2± 0.5

OTHER TEST CONDITIONS
- Photoperiod: 16h light/ 8h dark
- Light intensity: up to 500 lux
Reference substance (positive control):
not specified
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
other: biomass (fresh weight)
Details on results:
ANALYTICAL MEASUREMENTS, SECOND EXPERIMENT (nominal: 100 mg TiO2/L):
- 0 h after application of the test dispersion to the test medium, 110% of the nominal concentration was recovered.
- Within 1 d only 0.074 % of the nominal TiO2 concentration was detectable in the overlaying water phase.
- Until 28 d of exposure TiO2 concentration increased slightly to 1.55 % of the nominal value.
Reported statistics and error estimates:
Statistical evaluation of results
For evaluation of effects of the test substance on total number of worms after 28 days of exposure, Fisher’s Exact Binomial Test (multiple comparison, p ≤ 0.05, 1-sided greater) was used to determine significant differences in the mean number of worms between test concentrations and the control. Treatment means were compared by ANOVA followed by Dunnett’s test (multiple comparison, 1-sided smaller; p ≤ 0.05) and tested for statistically significant differences compared to the control. For evaluation of effects of NM-105 on the endpoints in the second test, the Student t test (pair-wise comparison, 1-sided smaller; p ≤ 0.05) was used for comparison with controls. All statistical calculations were done based on the nominal concentrations.
The statistical software package ToxRat Professional 2.10 (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

The particle size determined during exposure in the test media was between 402 and 1325 nm, declining over time.

Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the nano sized TiO2 material P25 (21 nm) were directly added to the overlaying water phase in a chronic sediment toxicity test according to OECD guideline 225. The derived 28-d NOEC values for biomass and reproduction of Lumbriculus variegatus were ≥ 100 mg/L (nominal). Chemical analysis confirmed that 110% of the nominal test concentration was applied to the overlaying water and that around 99% of the nominal concentration was removed from the water column after 1 d of exposure.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
TiO2 concentrations measured in exposure medium; Applied spacing factor of 10 and testing of only two concentrations; additional tests performed under food depleting conditions and/or performed with a too short exposure period of only 9 or 11 d; organic extract in test medium not described; light intensity not specified
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 202
Version / remarks:
1981 / 21 day reproduction test (including acute immobilization tests)
Principles of method if other than guideline:
Experiment 1a was performed according to OECD 202 (1981). Furthermore, additional experiments were performed which were not conducted according to OECD 202 (1981). These tests were performed under food depleting conditions (experiment 1b, 2b and 3b), or with a shorter test duration of only 9-11 d (experiment 2a/b and 3a/b). Hence, results of these studies are not considered for the hazard assessment of bulk TiO2. A spacing factor of 10 was used and only two concentrations were tested.
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
yes
Details on sampling:
Particles were dispersed in ASTM hard synthetic water (pH 7.8) following the described dispersion method. Thereafter, suspensions were left to settle for 24 h and water samples were taken.
Vehicle:
no
Details on test solutions:
- Direct addition of the test material into test vessel (dispersion): no
- Sonication of stock dispersion: Initial stock dispersions of particle suspensions of 1 g/L were dispersed in a ultrasonic bath during 30 min in MilliQ water and left for 24 h in agitation in darkness prior to be used to prepare test solutions (1 and 10 mg/L).
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: dispersions in ASTM medium were renewed every 24 h
- Test medium: ASTM hard synthetic water
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: clone F
- Feeding during test: yes
- Food type: C. vulgaris
- Amount:
150 µg C per daphnid per day (experiment 1a; experiment 2a and 3a)
30 µg C per daphnid per day (experiment 1b; experiment 2b and 3b)
- Frequency: daily
- photoperiod: 14 h light: 10 h dark
- temperature: 20 °C
- Light source: neon fluorescent lamp (UVA: < 10 µW/cm²)

ACCLIMATION
- Acclimation period: culture was maintained in ASTM hard synthetic water
- Type and amount of food: same as in test
- Feeding frequency: same as in test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
no information
Test temperature:
20 ± 1
pH:
7.8
Dissolved oxygen:
no information
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
Nominal (Tiona): 1 mg/L and 10 mg/L
Measured 24 h (Tiona): 0.61 ± 0.06 mg/L and 2.33 ± 0.31 mg/L, respectively
Details on test conditions:
TEST SYSTEM
- Test vessel:
Experiment 1: 120 ml screw top glass jars (6 cm water column)
Experiment 2 and 3: 1.2 L screw top glass vessels (16 cm water column)
- Aeration: no aeration
- Renewal rate of test solution (frequency): every 24 h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ASTM hard synthetic water with organic extract (not specified)
- Culture medium different from test medium: no, both ASTM hard synthetic water

OTHER TEST CONDITIONS
- pH: 7.8
- Photoperiod: 14 h light: 10 h dark
- Light intensity: UVA < 10 µW/cm²
- Light source: neon fluorescent lamps

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

TEST SET UP
- Experiment 1:
Food conditions: non-depleting (a) and depleting (b)
water column height: 6 cm
Shaking: no
Test duration: 21 d

- Experiment 2:
Food conditions: non-depleting (a) and depleting (b)
water column height: 16 cm
Shaking: no
Test duration: 9-11 d

- Experiment 3:
Food conditions: non-depleting (a) and depleting (b)
water column height: 16 cm
Shaking: yes, 2 rpm
Test duration: 9-11 d
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tiona AT 1
Basis for effect:
other: Age at first brood; Size of first brood; Size at first maturity; cummulative fecundity; body length at 21 d, population growth rate, body length at first maturity
Remarks on result:
other: Experiment 1a
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 2.33 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
Tiona At 1
Basis for effect:
other: Age at first brood; Size first brood; body length at first maturity; cummulative fecundity; body length at 21 d, population growth rate
Remarks on result:
other: Experiment 1a
Details on results:
Experiment 1b (tested under food depleting conditions):
No effects for Tiona were observed up to 10 mg/L.

Experiment 2a (1 L test vessels + non-depleting food conditions; test duration 9-11 d):
No effects for Tiona were observed up to 10 mg/L.

Experiment 2a (1 L test vessels + depleting food conditions; test duration 9-11 d):
Tiona significantly reduced brood size and population growth rate at 10 mg/L compared to control.

Experiment 3 a (1 L test vessels, non-depleting food conditions, resuspension):
No effects for Tiona were observed up to 10 mg/L.

Experiment 3b: (depleting food conditions; 1 L test vessels, resuspension):
No effects for Tiona were observed up to 10 mg/L.
Reported statistics and error estimates:
Each experiment and food ration was analyzed seperately by one way ANOVA to exclude confounding effects of inter-trail/experiment variation. When significant (P<0.05) treatment effects occurred, post hoc Tukey's or Dunnet's tests were performed to compare treatments. Prior to analyses all data except age at first reproduction were log tranformed to improve normality and vaiance homoscedasticity. Effects of treatment on age at first reproduction were analyzed by non-parametric Kruskal-Wallis test. Analyses of r were based on jackknife pseudovalues. Tests were conducted with the aid of the statistical package IBM SPSS v19.
Validity criteria fulfilled:
yes
Conclusions:
Dispersions (1 and 10 mg/L, nominal) of the TiO2 bulk material (Tiona AT 1, 200 nm, anatase) did not affect the age at first brood, size of first brood, cumulative fecundity, body length of adult females after 21 d and at first maturity, or the population growth rate of Daphnia magna in a semi-static standardized chronic toxicity tests according to OECD 202 (1981, and OECD 211). Derived 21 d-NOEC values for these endpoints amounted ≥ 10 mg/L (nominal) and ≥ 2.33 when based on measured concentrations of Tiona AT 1

Results of additional experiments are not relevant for the hazard assessment of TiO2, because they were either performed under depleting food conditions (experiments 1b, 2b and 3b), which are expected to enhance daphnia sensitivity, or were conducted with a shorter test period than that recommended in OECD 202 (1981, experiment 2a/b and 3a/b).

Description of key information

Dispersed microsized TiO2 is not chronically toxic to freshwater invertebrates, such as Daphnia magna, up to at least 10 mg/L (EC50: > 10 mg/L, nominal). Considering the low solubility of microsized TiO2, it is further concluded that microsized TiO2 is not toxic up to its solubility limit. Several guideline studies of dispersed nanosized TiO2 indicate that the chronic toxicity to aquatic freshwater invertebrates is also low with NOEC values ranging from > 1 mg/L to ≥ 100 mg/L.

Key value for chemical safety assessment

Additional information

Microsized TiO2:

Campos et al. (2013) tested dispersions (1 and 10 mg/L, nominal) of the TiO2 bulk material (Tiona AT 1, 200 nm, anatase) the age at first brood, size of first brood, cumulative fecundity, body length of adult females after 21 d and body length of adults at first maturity, and the population growth rate of Daphnia magna were not affected in a semi-static chronic toxicity tests according to OECD 202 (1981, and OECD 211). Derived unbounded 21 d NOEC values for these endpoints amounted ≥ 10 mg/L (nominal) and ≥ 2.33 when based on measured concentrations of Tiona AT 1. Results of additional non-standardised tests by Campos et al. (2013) are not relevant for the hazard assessment of TiO2 because the tests were performed under depleting food conditions, which are expected to enhance daphnia sensitivity, or for a shorter test period than recommended in OECD 211. One supporting study by Beim et al. (1982) on the chronic toxicity of microsized TiO2 to Daphnia magna reveals some toxic effects on the reproduction (30 d NOEC: ≥ 5-<10 mg/L) and survival (30 d NOEC: 70 mg/L) of daphnids. However, since limited information on test performance was provided, results from the study by Beim et al. (1982) are considered supporting data.

Furthermore, transformation/dissolution data of different microsized TiO2 materials indicate a low solubility in environmental media as dissolved Ti concentrations after 28 d were below the respective LOD/LOQ (< 0.11 / < 0.34 µg Ti/L). Hence, chronic invertebrate tests with dispersed microsized TiO2 were performed at TiO2 concentrations several magnitudes above the solubility limit. Thus, it is further concluded that microsized TiO2 is not chronically toxic to freshwater invertebrates up to its solubility limit.

Data on marine organisms does not exist.

Nanosized TiO2:

Campos et al. (2013) tested dispersions (1 and 10 mg/L, nominal) of the nanosized TiO2 materials NM 103 (20 nm, rutile, hydrophobic coating), NM 104 (20 nm, rutile, glycerin coating), and P25 (21 nm, rutile-anatase) and the age at first brood, size of first brood, cumulative fecundity, body length of adult females after 21 d, and the population growth rate of Daphnia magna were not affected in semi-static, chronic toxicity tests according to OECD 202 (1981); the respective 21 d NOEC value amounted to ≥ 10 mg/L (nominal) for all materials and ≥ 2.80, 3.12, and 2.70 mg/L when based on measured dispersed concentrations of NM 103, NM 104 and P25.
The body length of adults after 21 d was not affected at 10 mg/L NM 103, NM 104 or P25 compared to control organisms whereas the body length of adult organisms at first maturity was reduced at 10 mg P25/L (nominal). However, the endpoint body length of adults after 21 d is the standardised endpoint recommended in OECD 211 to assess effects on growth of D. magna. Furthermore, the observed effects of P25 on body length of adults at first maturity do not influence age at first brood, survival of adult organisms, and cumulative fecundity, and fitness measurements such as population growth rate, i.e. endpoints that are typically the most sensitive and also the most critical chronical endpoints according to OECD 211 and ECHA Guidance on IR & CSA, Chapter R. 7b (Version 3.0; February 2016). Thus, the respective 21 d NOECs of NM 103, NM 104 and P25 for reproduction and growth of Daphnia magna amount to ≥ 10 mg/L (nominal dispersed). Results of additional experiments performed in this study are not considered for the hazard assessment of nano-TiO2 because the tests were performed under depleting food conditions, which are expected to enhance daphnia sensitivity, or for a shorter test period than recommended in OECD 211.

Results of the study of Hund-Rinke et al. (2013) confirm that dispersed nano-TiO2 is not chronically toxic to daphnids up to 5 mg/L (nominal). In this study, daphnids were exposed to 1 and 5 mg/L (nominal) dispersed n-TiO2 (P25, 21 nm) in five independent semi-static limit tests (OECD 211) and the media were renewed daily or three times per week. Unbounded 21 d NOEC values of ≥ 5 mg/L (nominal) were determined for mortality, mobility and body length in all 5 experiments. Unbounded 21 d NOEC values of ≥ 5 mg/L for reproduction were derived in three of five experiments. However, reproduction was reduced by 10-16% at 5 mg/L in two experiments resulting in a LOEC of 5 mg/L (nominal). A NOEC should nevertheless not be derived from the latter two experiments since the separation factor should not exceed 3.2 according to OECD 211. Based on results of all five experiments by Hund-Rinke et al. (2013), the toxic potential of nano-TiO2 is low for daphnids with the NOEC being > 1 mg/L up to ≥ 5 mg/L. 

Evaluating the available data of all five tests by Hund-Rinke et al. (2013) and considering that Campos et al. (2013) did not observe effects on reproduction at concentrations of 10 mg/L n-TiO2 (nominal, dispersed), one may conclude that the long-term toxic potential of nano-TiO2 is low for daphnids.

Two studies on the chronic toxicity of nano-TiO2 to the epibenthic and endobenthic freshwater organisms Chironomus riparius and Lumbriculus variegatus exposed via the water phase, further indicate that the long-term toxic potential of nano-TiO2 is low for aquatic invertebrates. Although these tests are sediment tests, the organisms were exposed via the water phase so that the respective results are relevant for freshwater assessment. Schaefers et al. (2013) exposed Lumbriculus variegatus to dispersions of the nanosized TiO2 material P25 (21 nm) by direct addition to the overlaying water in a chronic sediment toxicity test according to OECD guideline 225. The derived 28 d NOEC values for biomass and reproduction of Lumbriculus variegatus were ≥ 100 mg/L (nominal). Furthermore, Hund-Rinke et al. (2013) tested the chronic toxicity of dispersions of the nanosized TiO2 materials P25 (anatase, rutile, 21 nm) and Homibkat UV 100 (anatase, 8 nm) applied to the overlaying water in a chronic sediment toxicity test according to OECD 219. Emergence and development rate of Chironomus riparius were not affected resulting in 28 d NOEC values of ≥ 100 mg n-TiO2/L (nominal). One supporting study on the chronic toxicity of nanosized TiO2 to the freshwater clam Corbicula fluminea additionally indicates that nanosized TiO2 is not chronically toxic up to 1 mg/L to freshwater invertebrates. However, only one concentration was tested and the organisms were not fed during the test so that results from this study are supporting data. 

Data for marine organisms are not available.

In sum, dispersions of microsized TiO2 material are not chronically toxic to freshwater invertebrates, such as Daphnia magna, up to 10 mg/L (nominal). Considering the low solubility of microsized TiO2, it is further concluded that microsized TiO2 is not chronically toxic up to its solubility limit.The long-term toxic potential of dispersed nano-TiO2 is also low for freshwater invertebrates with NOEC values ranging from > 1 mg/L to ≥ 100 mg/L.