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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 1991-December 1991
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: According to national guidelines. Methods and results generally well documented, but water characteristics and statistics not reported, no quantification of titanium concentration of test dispersions.
Qualifier:
according to
Guideline:
other: EPA-660/8-87/011, 1987
Deviations:
not specified
Qualifier:
according to
Guideline:
other: ASTM Standard E729 (1986)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): yes
Sonication/shaking or stirring of stock or test dispersion (duration): titanium dioxide was directly added into the test tube
Solvents: no
Filtration or other separation of test material from test dispersion: no
Appearance of dispersion: After 24 h all TiO2 settled out of the water column and formed a sediment on the bottom of the test chambers. Exception Tronox CRX, these TiO2 particles appeared to remain dispersed for 48h.
Renewal of test dispersions: no
test medium: well water
Test organisms (species):
Daphnia magna
Details on test organisms:
collected from Dr. Freida Taub at the University of Washington in Seatle and cultured using methods described by Goulden, et al. (1982)
fed with a mixture of vitamin enriched Ankistrodesmus falcatus, Selenastrum capricornutum and Chlamydomonas reinhardi 90
less than 24 hrs old
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Microscopic examination (10x) of the daphnia following exposure to TiO2, showed that the organisms ingested the materials and passed it trough the gut. Internal damage was not apparent in 48 hrs.
Test temperature:
20°C
Nominal and measured concentrations:
nominal exposure concentrations not described
Details on test conditions:
Well water passed through a treatment system containing limestone pH adjustment, Zeata Sol iron removal, carbon filtration and UV sterilization
Titanium dioxide was added directly into volumetric flasks
Two replicates per concentration contained 10 daphnia in a total of 100 ml of dispersion
16:8h light dark cycle
pH, conductivity, hardness measured at start end end of the test (data not shown).
Daphnia were gently touched with a pasteur pipet at 24 and 48 hrs. If the daphnia could not swim actively for 15 seconds immobilisation was recorded.
Nominal exposure concentrations not described
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Titanox 1000
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tronox CRX
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tioxide R-FC6
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tiona RCL-69
Basis for effect:
mobility
Details on results:
24h EC50: > 1000 mg/L
Reported statistics and error estimates:
EC50 computed using the probit analysis
Validity criteria fulfilled:
not specified
Conclusions:
Dispersions of four different micro sized TiO2 materials did not affect the mobility of Daphnia magna (< 24 h old) in an acute toxicity test according to US EPA (660/8-87/011; 1987) and ASTM (Standard E729, 1986) guidelines. 48 h-EC50 values of >1000 mg TiO2/L (nominal) were derived for all materials, including Tronox CRX (TiO2) which was described to have remained in the water phase for 48 h.
Endpoint:
short-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: OECD and US EPA guideline. Methods and results generally well documented, no quantification of titanium concentration of test dispersions.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
other: U.S. Environmental Protection Agency (660/3-75-009), 1975: Methods for Acute Toxicity Tests with Fish, Macro-invertebrates and Amphibians
GLP compliance:
not specified
Specific details on test material used for the study:
Titanox 1000: pigmentary titanium dioxide (particle size: 5-6 µm)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): yes
Sonication/shaking or stirring of stock or test dispersion (duration): no, test material was directly added to the test water
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of dispersion: no information
Test medium: Public drinking water passed through particle filters, activated charcoal filters an aged a minimum of 48 h in a 200 gallon polyethylene holding tank.
Test organisms (species):
Daphnia magna
Details on test organisms:
First instar Daphnia magna
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
50-65 ppm CaCO3
Test temperature:
20±1°C
pH:
6.8 - 7.0
Nominal and measured concentrations:
Nominal total, 1000 mg/l titanium dioxide.
Details on test conditions:
250 ml glass beakers containing 100 ml of sample.
Two replicates per treatment group
10 neonates/replicate
16:8 light-dark cycle
nominal exposure concentrations not described
pH, water hardness were determined during the test
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Titanox 1000
Basis for effect:
mobility
Validity criteria fulfilled:
not specified
Conclusions:
The dispersion of the micro sized TiO2 material Titanox 1000 (particle size: 5-6 µm) did not affect the mobility of first instar Daphnia magna in an acute toxicity test, which was performed according to OECD 202 and US EPA (660/8-87/011; 1987) guidelines. The determined 48 h-EC50 value was > 1000 mg TiO2/L (nominal).
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2007-02-05 to 2007-03-23
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Testing followed standardized guideline and conducted under GLP; Test material not further characterized; Exposure concentrations not measured
Qualifier:
according to
Guideline:
other: ISO 14669 (1999) Water quality-determination of acute lethal toxicity to marine copepods (copepoda:crustacea)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO 5667-16 (1998) Water quality sampling-guidance on biotesting of samples
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Not further specified.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): no
Sonication/shaking or stirring of stock or test solution/dispersion (duration): As floating and settled TiO2 particles were observed at 1000 mg/L in sea water, exposures were carried out with the supernatant of the TiO2 dispersions. Supernatants were prepared by the direct addition of the required nominal weights or volumes to seawater followed by gentle stirring for approximately 20 hours followed by a settling period of approximately one hour. After this settling period, the middle phase of the preparation was siphoned.
Solvents: no
Filtration or other separation of test material from test solution: If suspended material was observed then centrifugation (not specified) of the stocks was performed.
Appearance of solution: no information
Renewal of test dispersion: static, no
Test medium: Natural seawater (Scapa Flow, Orkney) is UV sterilised and filtered to 0.45 µm.
Test organisms (species):
other aquatic crustacea: Acartia tonsa
Details on test organisms:
- Source: obtained from age standardised cohorts, Dunstaffnage marine laboratory in 1995
- Age: 17-25 days old at definitive test commencement
- Strain: CCAP 1077/5
- Culturing: in accordance with SOP 501 ; culture system: flowing 0.45 µm filtered ultra violet treated sea water supplied by pump from scapa flow in Orkney
- Diet: mixed algal diet comprising of between 2 and 4 species.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 +/- 2°C
pH:
8.06-8.19
Dissolved oxygen:
88-94%
Salinity:
35 ‰
Nominal and measured concentrations:
10000, 5600, 3200, 1800, 1000 mg/L, plus four control vessels
Definitive test concentrations depend on the magnitude of response evident in the range finding test and employ five concentrations, plus four control vessels.
Details on test conditions:
TEST SYSTEM
- Control mortality: 5%


TEST MEDIUM / WATER PARAMETERS
- Dilution water: pH 8.00
- Stock solution:s floating and settled TiO2 particles were observed at 1000 mg/L in sea water, exposures were carried out with the supernatant of
the TiO2 dispersions. Supernatants were prepared by the direct addition of the required nominal weights or volumes to seawater followed by gentle stirring for approximately 20 hours followed by a settling period of approximately one hour. After this settling period, the middle phase of the preparation was siphoned, avoiding incorporation of undissolved particles, if present. If suspended material was observed then centrifugation of the stocks was performed.
- A reference test was conducted using 3,5 Dichlorophenol at 0.32, 1.0, 1.8 and 3.2 mg.l-1.
- Natural seawater is supplied by a pump from Scapa Flow, Orkney. All seawater is UV sterilised and filtered to 0.45 µm.



TEST PROCEDURE
- in 100 ml capacity borosilicate glass crystallising dishes, each containing 50 ml of test medium covered with soda glass watch covers.
- In the Rangefinding tests, five animals were exposed per concentration and in definitive tests ten animals were exposed per replicate (20 per treatment).
- Test animals were transferred to test vessels using glass pipettes of three to five millimetres in diameter (internal) and with fire polished rims.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
in supernatant (siphoned after 1 h of sedimentation following 20 h of stirring)
Basis for effect:
mortality
Results with reference substance (positive control):
48 h-LC50: 1.26 mg/L (informal reference range: 48 h-LC50 0.4-1.5 mg/L)
Reported statistics and error estimates:
Mortalities are determined in each vessel at 24 and 48h. The number of dead or immobile copepods are expressed as a proportion of the total number exposed.
Where sufficient response is observed, the 24 and 48h LC50, 48h LC90 and 48h NOEC values are calculated using an appropriate statistical method from the ToxCalc Version 5 software.

LC50 24h: >10000 mg/l

LC90 48h: >10000 mg/l

NOEC 48h: >= 10000 mg/l

Validity criteria fulfilled:
yes
Remarks:
test was conducted on accordance with the study plan and met all relevant validity criteria
Conclusions:
Supernatants which were collected from a TiO2 dispersion after 24 h of stirring followed by 1 h of sedimentation, did not affect the survival of the 17-25 d old marine crustacean Acartia tonsa in an acute toxicity test according to ISO 14669 (1999), and a 48 h-LC50 value of > 10,000 mg/L (nominal) was reported. Test material not specified and exposure concentration not measured.
Endpoint:
short-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: see 'Remark'
Remarks:
short communication, hence very little information given on methodology and study performance, suspension tested and total titanium concentration measured; However, used ICP-OES method and sampling method was not further described; acclimatisation period not specified; test material was incorrectly tested: environmentally not relevant, high concentrations were tested, causing a physical attachment of the particles to the appendages of daphnids, limiting their mobility; natural test medium was not analyzed for chlorine and pseticede content
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): no
Sonication of dispersion: dispersions were sonicated for 30 min in a water bath (no further information)
Solvents: no
Filtration or other separation of test material form test dispersion: no
Appearance of dispersion: sedimentation of particles occured
Renewal of test dispersion: no (static)
Test medium: ISO test water and real river water
Test organisms (species):
Daphnia magna
Details on test organisms:
No information
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
no data
Test temperature:
no data
pH:
ISO test water: 7.54
real river water: 8.15
Dissolved oxygen:
ISO test water: 6.78 mg/L
real river water: 7.52 mg/L
Salinity:
not relevant
Conductivity:
ISO test water: 0.5 mS/cm
real river water: 0.23 mS/cm
Nominal and measured concentrations:
nominal concentration: 0 (control), 10, 18, 32, 58, and 105 mg/L TiO2
measured concentrations: Ranges of measured concentrations reported, shown concentration response relationships were based on measured concentrations; However, time point of measurements not reported, EC50 values based on measured concentrations.
Details on test conditions:
TEST SYSTEM
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- Test performance: in the dark

TEST MEDIUM / WATER PARAMETERS
Source/preparation of dilution water:
- Conductivity: (ISO water) 0.58 mS/cm; (river water) 0.23 mS/cm
- Total organic carbon: (river water) 1.5 mg/L
- Metals: (river water) Zn2+, Pb2+, Ni2+, Hg2+, Cu2+, Cr3+, and Cd2+ were below the instrumental limit of detection, 5µg/L
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Remarks:
ISO test water
Effect conc.:
29.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25
Basis for effect:
mobility
Remarks on result:
other: CL (95%): 16.8 - 53
Duration:
48 h
Dose descriptor:
EC50
Remarks:
ISO test water
Effect conc.:
19.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25
Basis for effect:
mobility
Remarks on result:
other: CL (95%): 9.9 - 37.7
Remarks:
Measurement time point unknown, consequently value considered as not reliable
Duration:
48 h
Dose descriptor:
EC50
Remarks:
river water
Effect conc.:
33.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25
Basis for effect:
mobility
Remarks on result:
other: CL (95%): 22.8 - 44.4
Duration:
48 h
Dose descriptor:
EC50
Remarks:
river water
Effect conc.:
27.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25
Basis for effect:
mobility
Remarks on result:
other: CL (95%): 19.0 - 36.7
Remarks:
Measurement time point unknown, consequently value considered as not reliable
Details on results:
At high NP concentration (10-100 mg/L), it was observed that the TiO2 was highly attached to the daphnids’ appendages, limiting their mobility. It is discussed that in this case, a physical impediment to swimming may be the toxicity mechanism.

No information on water sampling method for TiO2 analysis reported. Considering, that turbidity was decreased by 87.9% and 97.5% in ISO and river water after 48 h, the reported TiO2 concentrations, which correspond to approximately 64-80% of nominal values, do not represent TiO2 concentrations after 48 h. Consequently, the effect values based on measured concentrations are not considered reliable.
Reported statistics and error estimates:
not reported
Validity criteria fulfilled:
not specified
Conclusions:
Dispersions of a nano sized TiO2 material (P25) in ISO test water and river water inhibited the mobility of Daphnia magna (<24 h old) under dark conditions in an acute toxicity test according to OECD 202 guideline. Inhibition of mobility was more pronounced in ISO test water (48 h-EC50: 29.7 mg/L, nominal) than in real river water dispersions (48 h-EC50: 33.6 mg/L, nominal). Nevertheless, at TiO2 concentrations between 10-100 mg/L “the TiO2 was highly attached to the daphnids’ appendages, limiting their mobility” and being “a physical impediment to swimming” and possibly particle attachment resulted in a “blockage of gills”. This means that the derived EC50 values were based on mechanical effects and not on intrinsic properties of the test substance and thus cannot be used for the environmental hazard assessment of nano sized TiO2.
Endpoint:
short-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: no quantification of titanium concentration of test dispersions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Direct addition of test material into test vessel (dispersion): no
- Sonication of dispersions: A stock suspension of 20% (m/v) was prepared by dispersing appropriate amounts of TiO2 NP powder in deionized water, vortexed for 20 s, and then sonicated (ultrasonic bath) for about 10 min for maximum dispersion. Appropriate volumes of the stock suspension were then immediately transferred into the exposure tanks containing Artemia nauplii or adults in the seawater.
- Solvents: no
- Filtration or other searation of test material form test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: no
- Test medium: Seawater (30‰ m/v)
Test organisms (species):
Artemia salina
Details on test organisms:
TEST ORGANISM
- Common name: Artemia salina
- Source: Artemia cysts (The Great Salt Lake, Utah harvest) from Artemia International LLC, Houston TX, kept at 4°C temperature moisture-free container in a refrigerator
- Age at study initiation: nauplii + adults
- Feeding during test: no food was provided during the course of the exposure

PREPARATION OF TEST ORGANISM
Artemia cysts were hatched (procedure described by Persoone et al., 1989) in seawater (30‰ m/v) within 24 h:
- cysts were prepared by dissolving appropriate amount of Instant Ocean® salt in deionized water, stirred for 24 h under aeration and then filtered through 30-μm Millipore cellulose filters.
- encysted Artemia: hydrated in distilled water at 4 °C (12 h) and then washed to separate the floating cysts from those that sink (collected on a Buchner funnel and washed with cold deionized water).
- Approximately 3 g of the pre-cleaned cysts were incubated in 1.5 L seawater in a conical plastic contained with graduations at 30 ± 1 °C.
- Aeration by a small line extending to the bottom of the hatching device from an aquarium air-pump.
Counting hatched Artemia:
- procedure described by Sorgeloos (1980); 100 mL solution containing Artemia nauplii into a clean beaker. Under continuous stirring, 1 mL of this stock was diluted to 100 mL with seawater (100-fold dilution). Next, 0.1 mL of the diluted solution taken under stirring and placed in petridish. The number of nauplii was determined by counting visually in this volume (0.1 mL).
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
acute toxicity conducted for 24 and 96h
Hardness:
no information
Test temperature:
24 ± 2 °C
pH:
8.1 - 8.7 (measured at the beginning and at the end of exposure)
Dissolved oxygen:
no information
Salinity:
26 ppt (Instant Ocean® salt: 30 g/L)
Conductivity:
no information
Nominal and measured concentrations:
nominal concentration: 0, 10, 50, 100 mg/L TiO2 NPs
Details on test conditions:
TEST MEDIUM:
- seawater (30‰ m/v), Instant Ocean® salt applied to deionized water, stirred for 24 h under aeration and then filtered through 30-μm Millipore cellulose filters

TEST SYSTEM:
- 24 h and 96 h (Artemia nauplii and adults)
- Three different test concentrations (10, 50 and 100 mg/L) of TiO2 NPs
- triplicate measurements in conical plastic containers (1-L and 2-L inner volume)
- Exposures in 500 mL and 1500 mL seawater (Artemia nauplii and adults, respectively)
- Aeration by a line extending to the bottom of the conical flask to prevent settling of NPs from suspension during the course of the exposure
- 1500 lux day-light (by a fluorescent lamp; 16:8 h light:dark).

Preliminary exposure studies were conducted with up to 5 mg/L TiO2 NPs on Artemia nauplii to estimate the exposure concentration. No significant immobilization or mortality occurred within 24 h; therefore, the experimental NP suspensions were prepared between 10 and 100 mg/L to achieve measurable effects.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
suspension
Basis for effect:
mortality
Remarks:
Artemia nauplii
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
suspension
Basis for effect:
mortality
Remarks:
Artemia adults
Details on results:

Effect of concentration of TiO2 NP suspension on mortality (toxicity):
- aqueous suspensions of TiO2 NPs were not acutely toxic to Artemia at elevated levels (100 mg/L), even to the most vulnerable nauplii (LC50 > 100 mg/L).

Effect of exposure time on mortality:
- In 24 h, treatments exhibited similar rate of mortality to that of controls, but the values increased significantly in 96 h.
- mortality increased from 6 to 18% for nauplii and 5 to 14% for adults at 100 mg/L TiO2 NP suspension (LC50 > 100 mg/L).

Stability of TiO2 in water:
- Aggregation was minimal in freshly prepared stock suspension; size: 8 - 40 nm
- DLS: instantaneous aggregation in water; hydrodynamic sizesfrom 210 to 1833 nm; estimated mean size: 371, 498, and 589 nm for 10, 50 and 100 mg/L TiO2 NPs, respectively. The size of aggregates tended to increase with TiO2 concentration.
- TEM: The median size of the aggregates increased from 580.5 nm to 2349 nm and then to 3526 nm within 1, 12 and 24 h, respectively.

Elimination of ingested TiO2 NPs:
- similar elimination patterns: 3 - 12% reduction of Ti levels
- The TEM and DLS data clearly show that TiO2 NPs were no longer nanometer size particles but aggregates in the exposure medium. Nevertheless, Artemia, even nauplii, accumulated the aggregates from water readily within 24 h.

Oxidative stress induced by suspensions of TiO2 NPs:
- suspensions of TiO2 NPs were totally benign to Artemia in 24 h.
- No significant toxicity was observed from any of the suspensions to nauplii or adults.
- In 96 h, the MDA concentrations in treatments increased compared with the controls that substantiated that prolonged exposure induced oxidative stress on Artemia. In addition, the MDA levels closely correlated with the mortality rates (r2 > 0.9 for nauplii and adults) indicating that the mortalities were due to the oxidative stress (caused by the deprivation from food or starvation rather than the chemical toxicity of the suspensions of TiO2 NPs).
Reported statistics and error estimates:
- All experiments were repeated three times independently.
- Data were recorded as the mean with standard deviation (SD).
- One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons to detect significant differences in mortality and accumulation rates among the controls and treatments.
- In all data analyses, a p-value of 0.05 was considered statistically significant.
Validity criteria fulfilled:
not specified
Conclusions:
In this study, the acute toxicity of nano sized TiO2 dispersions (10-30 nm) to Artemia salina (nauplii and adults) were investigated after 24 h and 96 h according to the OECD 202 test guideline. No significant mortality or toxicity occurred at any dose, resulting in 96 h-LC50 > 100 mg/L (nominal). Exposure concentrations not measured.
Endpoint:
short-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: no quantification of titanium concentration of test dispersions; only sedimentation monitored over the exposure period; light cycle not defined; additional tests were performed under non-standardized light conditions with UVA and UVB irradiation
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
Photoperiod not specified.
Principles of method if other than guideline:
Tests were either performed under visible light (> 400 nm), or under non-standardized light conditions with UVA or UVB irradiation.
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition of test material into test vessel (dispersion): no
- Sonication of stock (duration): yes, 10 minutes with a high frequency probe at 120 W/L
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of solution: no information
- Renewal of test solution: yes on daily basis
- Test medium: 3% saline solution (Red Sea Salt) prepared in destilled water


Stock suspensions (1 g/L) were prepared in culture medium by sonication for 10 min. Immediately after the sonification, dilutions of 1, 10 and 100 mg/L were prepared.
Test organisms (species):
Artemia salina
Details on test organisms:
TEST ORGANISM
- Common name: brine shrimp
- Source: Nauplii were obtained after 48-h incubation of commercial cysts (Maramar, Brazil)
- Feeding during test: no

Test type:
semi-static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
not stated
Test temperature:
20 ± 1 °C
pH:
8 ± 1
Dissolved oxygen:
6 ± 0.5 mg/L
Salinity:
3%
Conductivity:
40 ± 1 µS/cm
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 166, 500 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Petri dishes 5 cm diameter
- Material, size, headspace, fill volume: glass, 10 mL
- Renewal rate of test solution (frequency/flow rate): renewal after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water, filtered with activated carbon

OTHER TEST CONDITIONS
- Photoperiod: not stated
- Light intensity: 250 ± 79 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- mobility
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
284.81 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Combined exposure of n-TiO2 and UV light lead to an 48-h EC50 of 4.03 mg/L.However, tests were performed under non-standard light conditions and are therefore considered not relevant for regulatory hazard assessment purposes.
Reported statistics and error estimates:
The EC50 values as well as the corresponding 95% confidence intervals were calculated by probit analysis (Statgraphics Plus v.5.1 software)
Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the nano sized TiO2 material Aeroxide P25 (particle size: 25 nm) affected the mobility of Artemia salina nauplii in an actue immobilisation test (similar to OECD 202). The determined 48-h EC50 was 284.81 mg TiO2 NP/L (nominal) under laboratory light conditions.
Pronounced toxicity was found when organisms were exposed to P25 under non-standard light conditions with UVA or UVB irradiation.
Endpoint:
short-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: no quantification of titanium concentration; only sedimentation monitored; light cycle not mentioned; additional tests were performed under non-standardized light conditions with UVA and UVB irradiation
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
Photoperiod not specified.
Principles of method if other than guideline:
Tests were either performed under visible light (> 400 nm), or under non-standardized light conditions with UVA or UVB irradiation.
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition of test material into test vessel (dispersion): no
- Sonication of stock (duration): yes, 10 minutes with a high frequency probe at 120 W/L
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of solution: no information
- Renewal of test solution: yes on daily basis
- Test medium: Tap water

Stock suspensions (1 g/L) were prepared in culture medium by sonication for 10 min. Immediately after the sonification, dilutions of 1, 10 and 100 mg/L were prepared.
Test organisms (species):
other: Daphnia similis
Details on test organisms:
TEST ORGANISM
- Common name: waterflea
- Source: not stated
- Age at test initiation: neonates
- Feeding during test: no


Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2° dGH
Test temperature:
20 ± 1 °C
pH:
7.9 ± 1
Dissolved oxygen:
6 ± 0.5 mg/L
Salinity:
not stated
Conductivity:
133 ± 1 µS/cm
Nominal and measured concentrations:
Nominal concentrations of 0 (control), 100, 1000 mg/L were used under standard labotory light conditions.
Details on test conditions:
TEST SYSTEM
- Test vessel: Petri dishes 5 cm diameter
- Material, size, headspace, fill volume: glass, 10 mL
- Renewal rate of test solution (frequency/flow rate): renewal after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water, filtered with activated carbon

OTHER TEST CONDITIONS
- Photoperiod: not stated
- Light intensity: 250 ± 79 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- mobility

Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Combined exposure of n-TiO2 and UV light lead to an 48-h EC50 of 60.16 mg/L. However, tests were performed under non-standard light conditions and are therefore considered not relevant for regulatory hazard assessment purposes.
Reported statistics and error estimates:
The EC50 values as well as the corresponding 95% confidence intervals were calculated by probit analysis (Statgraphics Plus v.5.1 software)
Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the nano sized TiO2 material Aeroxide P25 (particle size: 25 nm) did not affect the mobility of Daphnia similis neonates in an actue immobilisation test (according to OECD 202) up to the highest test concentration. The determined 48-h EC50 was > 1000 mg TiO2 NP/L (nominal) under laboratory light conditions. Pronounced toxicity was found when organisms were exposed to P25 under non-standard light conditions with UVA or UVB irradiation.
Endpoint:
short-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: well documented study according to ASTM guidelines, no quantification of titanium concentration of test dispersions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: American Society for Testing and Materials: Standard guide for conducting acute toxicity tests on test materials with fishes, macro invertebrates and amphibians.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- Direct addition of test material into test vessel (dispersion): no
- Sonication of dispersions: 10 mg of nanometal powder was added to 10 ml of ultrapure Milli-Q water (Millipore, Billerica, MA, USA) and then sonicating with a probe sonicator for six halfsecond pulses at an output of 6 W and 22.5 kHz. Preparation immediately before test start.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: unclear, only described as static renewal conditions
- Test medium: moderately hard freshwater
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
- Neonates, less than 24 h old.
- Before the start of each test, the neonates were separated from the adult daphnids and fed a mixtures of 7 ml of YCT (yeast, cerophyll, trout chow) and 7 ml of algae (P. subcapitat) for every liter of invertebrate culture.
Test type:
semi-static
Water media type:
freshwater
Remarks:
static renewal period duration not described
Limit test:
no
Total exposure duration:
48 h
Hardness:
Test water: 142 ± 2 mg/l as CaCO3
Test temperature:
20 ± 2 °C
pH:
Test water: 8.2 ± 1
Dissolved oxygen:
Test water: 8.5 - 8.9 mg/l
Conductivity:
395 µS
Nominal and measured concentrations:
nominal, up to 10 mg/L, no further description
Details on test conditions:
- 10 neonates were transferred to each test container (30-ml plastic cups) using a small, wide-mouth, plastic pipette to minimize the transfer of culture water. The sample dilutions, if necessary, were prepared with filtered test water, and 20 ml of the sample or its dilutions were added to cups containing the neonates.
- Each concentration contained four replicate exposures.
- five concentrations tested
- Negative controls were performed simultaneously (filtered test water was used).
- Ambient lighting.
- 20 °C
- static renewal exposure (not further described)
- endpoint: mobility
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
mobility
Reported statistics and error estimates:
LC50 values were calculated using the trimmed Spearman-Karber method. Control survival was required to exceed 90%; if control survival was less
than 90%, the test was repeated.
Validity criteria fulfilled:
not specified
Conclusions:
Dispersions of the TiO2 nanomaterial P25 (20 ± 6.7 nm) did not affect the mobility of Cerodaphnia dubia (age < 24 h old) in an acute toxicity test according to ASTM standard guidance for macro invertebrates under semi-static test conditions. The observed 48 h-EC50 value was > 10 mg/L (nominal). Exposure concentrations not measured.
Endpoint:
short-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: well documented study according to ASTM guidelines, no quantification of titanium concentration of test dispersions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: American Society for Testing and Materials: Standard guide for conducting acute toxicity tests on test materials with fishes, macro invertebrates and amphibians.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- Direct addition of test material into test vessel (dispersion): no
- Sonication of dispersions: 10 mg of nanometal powder was added to 10 ml of ultrapure Milli-Q water (Millipore, Billerica, MA, USA) and then sonicating with a probe sonicator for six halfsecond pulses at an output of 6 W and 22.5 kHz. Preparation immediately before test start.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: unclear, only described as static renewal conditions
- Test medium: moderately hard freshwater
Test organisms (species):
Daphnia pulex
Details on test organisms:
Cultures were maintained continuously for six months, and daphnids were fed YCT (yeast, cerophyll, trout chow) media ad libitum daily before use.
Not fed during testiong.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Test water: 142 ± 2 mg/l as CaCO3
Test temperature:
25 °C
pH:
Test water: 8.2 ± 1
Dissolved oxygen:
Test water: 8.5 - 8.9 mg/l
Conductivity:
395 µS
Nominal and measured concentrations:
nominal, up to 10 mg/L, no further description
Details on test conditions:
- Five adult daphnids (age not described) were placed into 500 ml beakers containing 200 ml of test solution in filtered (pore size, 0.45 µm) test water.
- Each concentration contained four replicate exposures.
- Five concentrations tested per treatment
- Negative controls were performed simultaneously.
- 25 °C
- 14:10h light/dark
- Beakers were monitored daily and dead individuals were removed. Death was assessed by lack of movement or response to gentle prodding.
- no feeding during the test
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25
Basis for effect:
mobility
Reported statistics and error estimates:
LC50 values were calculated using the trimmed Spearman-Karber method. Only tests with < 10% immobility of control daphnids after 48 h were considered valid.
Validity criteria fulfilled:
not specified
Conclusions:
Dispersions of the TiO2 nanomaterial P25 (20 ± 6.7 nm) did not affect the mobility of adult Daphnia pulex in an acute toxicity test according to ASTM standard guidance for macro invertebrates under semi-static test conditions. The observed 48 h-EC50 value was > 10 mg/L (nominal). Exposure concentrations not measured.
Endpoint:
short-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: Study well performed and doucmented; Initially applied total TiO2 concentration not measured
Qualifier:
no guideline followed
Version / remarks:
However, study well described
Principles of method if other than guideline:
TiO2 application via the water phase.
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
yes
Details on sampling:
- Sampling of the water phase after 0.5, 1, 2, 3, 20, 24, 27 and 44 h of exposure
Vehicle:
no
Details on test solutions:
- Direct addition of test material into test vessel (dispersion): no
- Sonication of dispersions: A stock suspension (1000 mg/L)was prepared by sonication in Lake Superior water for 1 h in a bath-type sonicator (35 kHz frequency; Fisher Scientific). Stock dispersions were sonicated for 30 min prior to making working suspensions. Working suspensions were diluted from stock dispersions with the Lake Superior water and stirred for 20 min before the initiation of the bioassays.
- Solvents: no
- Filtration or other separation of test material form test dispersion: no
- Renewal of test disperions: no
- Test medium: Lake Superior water (LSW, St. Louis County, MN, USA) analyzed by Era Laboratories, Inc. (Duluth,MN,USA) for general characteristics (pH; hardness; alkalinity; conductivity; dissolved oxygen (DO); dissolved organic carbon (DOC); and chloride content. 16.57 mg/L NaCl was added in LSW for improving the survival of H. azteca.
Test organisms (species):
other aquatic crustacea: Hyalella azteca
Details on test organisms:
TEST ORGANISM
- Common name: Hyalella azteca
- Source: U.S. Environmental Protection Agency's (U.S. EPA's) Mid-Continent Ecology Laboratory, Duluth, MN, USA.
- Age at study initiation: adult and juveniles (7-8 d-old) cultures
- Neonates of 0–1-d-old were collected and added to 2-L beakers of Lake Superior Water (LSW) and maintained for 7 d.
- Adults and neonates were cultured at 23 °C. Photoperiod was maintained at 16L/8D with an illumination of about 1280 lx.
- Daily food: a 1:1:1 mixture of yeast:ceropyl:tetramin (YCT) and Pseudokirchneriella subcapitata.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
44 mg/L as CaCO3
Test temperature:
23 ± 1 °C
pH:
7.89 – 8.43
Dissolved oxygen:
7.8 - 8.6 mg/L
Salinity:
--
Conductivity:
10.5 mS/m
Nominal and measured concentrations:
- nominal concentrations (laboratory light): 0, 10, 50, 100, 250, 500, 750 and 1000 mg/L nano-TiO2
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker (50-mL)
- No. of organisms per vessel: 6
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5
- No test dispersion renewal

OTHER TEST CONDITIONS
- Photoperiod: 16h:8h (light/dark)
- Light intensity: laboratory light
- General characteristics: pH, 8; hardness, 44 mg/L as CaCO3; alkalinity, 44 mg/L as CaCO3; conductivity, 10.5 mS/m; dissolved oxygen (DO), 8.5 mg/L; dissolved organic carbon (DOC), 1.2 mg/L; and chloride, 1.8 mg/L.
- For bioassays, water quality parameters were monitored every 24 h throughout all experiments.
- All test solutions were kept at 23 ± 1 °C during the whole exposure process.
- Each beaker (50-mL) had 6 H. azteca, 1 mL silica sand, 30 mL Lake Superior Water (LSW), and 16.57 mg/L NaCl (for improving the survival of H. azteca)
- Aluminum-wrapped 50-mL beakers, stainless steel holder (L × W × h: 54 cm × 28 cm × 9 cm), glass tray with water (heat isolator, L × W × h: 55 cm × 30 cm × 2.5 cm), stainless steel screen, and neutral-density screen filters (L × W: 55 cm × 30 cm). Sidewalls of the stainless steel holder and the glass tray were wrapped with aluminum foil.
- Nano-TiO2 suspensions were added in each beaker 3 h ahead of the addition of organisms.
- The mortality of H. Azteca was recorded at the test termination.
- A preliminary study indicated that addition of 0.3 mL P. subcapitata (3.71 × 107 cells/mL) every day in each beaker led to 95%–100% survival of H. azteca.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
631 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE® TiO2 P 25
Basis for effect:
mortality
Remarks on result:
other: 95% CI; 427-933 mg/L
Details on results:
- LC50 (96 h): 631 mg/L (nominal); 95% confidence interval (CI), 427–933 mg/L (laboratory light)
- majority of the nano-TiO2 existed as agglomerates in all tests rather than discrete particles
- In the study, nano-TiO2 formed large agglomerates in less than 0.5 h in a natural water system, due to the presence of algae, high pH, moderate ionic strength, and high concentration of divalent cations.
- LC50 values calculated based on initial nano-TiO2 concentrations in the water column
- However, organisms were most probably exposed to even higher concentrations, because UV Vis spectroscopy showed a sedimentation of greater than 77%-99% of TiO2 particles after 0.5 h, resulting in TiO2 bottom concentrations higher than the initial TiO2 water concentrations.
- H. azteca, which is a benthic organism is most likely exposed to the sedimented TiO2 part within this exposure system.
Reported statistics and error estimates:
Median lethal concentrations (LC50s) and associated 95% confidence intervals (95% CI) were estimated from a tolerance distribution analysis using a three-parameter probitmodel (TRAP; Toxicity Relationship Analysis Program, v. 2.21, U.S. EPA). No limited partial effects were observed for all exposure series.
Validity criteria fulfilled:
yes
Conclusions:
In a well described 96-h acute bioassay following no specific test guideline, dispersions of the nanosized TiO2 material P25 (25.1 ± 8.2 nm) showed minimal toxicity to juvenile Hyalella azteca (age, 7-8 d) under laboratory light conditions, indicated by the median 96 h-LC50 of 631 mg/L (nominal). Measurements of TiO2 water concentrations emphasized a pronounced sedimentation of greater than 77% of the nominal TiO2 amount, indicating that these benthic organisms were exposed to bottom TiO2 concentrations exceeding the nominal water TiO2 concentrations. Furthermore, it is expected that these high concentrations lead to particle attachment, which might possibly result in a blockage of the gills of H. azteca. This means that the derived LC50 values were based on mechanical effects and not on intrinsic properties of the test substance and thus effect values will not be applied for CSA.
Endpoint:
short-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: No quantification of titanium concentration of test solutions, widely spaced (separation factor of 10) concentrations of the test substance; M4 medium contains EDTA
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): no
Sonication of stock suspension:
In total, 23 acute toxicity tests were run with different TiO2 materials. For all tests, a stock dispersion of 100 mg/L was prepared. In order to compare possible effects on daphnids caused by different dispersibility characteristics of the test compounds, different treatments of the stock dispersions were applied.

Uncoated nano-scale TiO2:
A) 5 min ultrasonication after transferring the test item into the test medium (M4, PW, SW)
B) 10 min stirring (PW)
C) 10 min stirring + quartz sediment (PW)
D) 5 min ultrasonication after transferring the test item into the test medium + quartz sediment (PW)
E) Wetted with H20 bidest*, 5 min ultrasonication
F) Wetted with H20 bidest, 2 h ultrasonication
G) Wetted with H20 bidest, 2 h ultrasonication, 0.2 µm filtered
H) Wetted with H20 bidest, 0.2 µm filtered
I) 48 h aging of test media (with re-dispersion)
J) 30 min daylight lamp (20W/m2, 300–800 nm; Hönle SOL 500 with UV-B filter, Munich, Germany)

* In order to achieve better wetting properties a concentrated stock dispersion of 1.11 g/L using demineralized water and ultrasonication for 5 min was generated. Thereafter, the stock dispersion was diluted with demineralized water to 111 mg/L, 11mg/L, etc. The resulting dilutions were diluted once more with 10-fold concentrated M4 medium at the ratio 1:10 in order to result in the final nominal concentrations of 0.01–100 mg/L.

T-Lite TM SF-S (coated):
A) 5 min ultrasonication (M4, PW, SW)

T-Lite TM SF (cated):
A) 20 h stirring and 5 min ultrasonication (M4)

T-LiteTM MAX (coated):
A) 20 h stirring and 5 min ultrasonication

Solvents: no
Filtration or other seperation of test material from test dispersion: see G) and H) other experiments: no
Appearance of dispersion: In all tests, except those using filtered test medium, the test compounds were visually observed to not completely disperse at concentration levels 10–100 mg/L. The dispersions appeared turbid and settled material was observed at the bottom of the test vessels after 24-h and 48-h. During the test periods further sedimentation of particles was visually observed.
renewal of test dispersion: no (static)
Test medium: Three different test media were used: Artificial Elendt M4 medium (M4), pond water sieved with 55 µm sieve (PW), well-spring water of the River Selz in south-western Germany (SW)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: D. magna Strauss
- Source: laboratory stock culture at the test facility
- Age at study initiation (mean and range, SD): <=24 h old
- Food type: suspension of the green algae Desmodesmus subspicatus
- Frequency: daily
- Organisms were not fed during the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2.02 - 2.86 mmol/L M4 and PW
5.95 mmol/L SW
Test temperature:
20±1 °C
pH:
7.1 - 8.7
Dissolved oxygen:
> 3 mg/L
Salinity:
not relevant
Conductivity:
M4: 577-605 µS/cm²
PW: 752-879 µS/cm²
SW: 1108 µS/cm²
Nominal and measured concentrations:
nominal concentrations: 4-5 concentrations (0.1-100 mg/L or 0.01-100 mg/L) graduated by a factor of 10 and untreated controls without the test compound
Details on test conditions:
TEST SYSTEM
- Test vessel: glass tubes
- Material, size, headspace, fill volume: 10 mL
- Renewal rate of test solution (frequency/flow rate): static
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 1

- Test concentration: max. 5 (from 0.01-100 mg/L); factor: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 (according to OECD 202)
- Source/preparation of dilution water: PW pond water-plankton was separated by straining the water through a 55 µm sieve
- Source/preparation of dilution water: SW river selz, southwest Germany
- Total organic carbon: 3.5–7.8 mg/L PW
- Total organic carbon: 1.3 mg/L SW
- Conductivity: 577 - 605 µS/cm M4
- Conductivity: 752 - 879 µS/cm PW
- Conductivity: 1108 µS/cm SW
- TOC PW: 3.5-7.8 mg/L
- TOC SW: 1.3 mg/L

Experiments, which were performed in M4 medium are not considered to be relevant for hazard assessment purposes of nano-TiO2, because M4 medium contained EDTA.

Oxygen content and pH of the test media were measured at the beginning and at the end of the test in one replicate per control and per test concentration level (Multiline P4, WTW, Weilheim, Germany).

OTHER TEST CONDITIONS
- Photoperiod: 16h light/ 8h dark
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
for all test materials for all tests
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
3.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
uncoated nano-scale TiO2
Basis for effect:
mobility
Remarks:
; test A), SW
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
uncoated nano-scale TiO2
Basis for effect:
mobility
Remarks:
; all other tests with uncoated nano-TiO2
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
T-Lite SF-S (coated)
Basis for effect:
mobility
Remarks:
; PW, SW
Results with reference substance (positive control):
Reference acute toxicity tests using potassium dichromate were performed regularly at intervals of 1–2 months, whereby the median effective concentration (EC50) after 24 h ranged from 0.6 to 1.4 mg/L during the last 10 months, thus meeting the validity criterion according to the OECD guideline.
Reported statistics and error estimates:
Effective concentrations (EC10 and EC50 values) for the parameter immobilization were calculated with the help of the results obtained from the 48-h acute toxicity tests. The dose–response curve was fitted via the probit model according to Finney (1971). If the goodness-of-fit test Pearson Chi-Square was significant (p 6 0.05), high or low doses were excluded from the evaluation. This analysis was only performed, if interpolation was possible. In those cases were extrapolation was necessary the EC10 or EC50 were given as >100 mg/L. All calculations were done withthe help of Microsoft Excel 2000.

Tests in M4 medium:

for all test materials in M4 medium:

- 48 h-EC50 (mobility): > 100 mg/L (nominal)

T-Lite Max (coated; test A)

- 48 h-EC10 (mobility): 76.4 mg/L (nominal)

nano-scale TiO2 (uncoated; test A)

- 48 h-EC10 (mobility): > 85.1 mg/L (nominal)

T-Lite SF-S (coated)

- 48 h-EC10 (mobility): >100 mg/L (nominal)

Validity criteria fulfilled:
yes
Remarks:
according to OECD guideline
Conclusions:
Daphnia magna (< 24 h old) were exposed to dispersions of four nano-TiO2 materials differing in crystalline phase, size, and coating, which were prepared with different dispersion techniques using either artificial or natural waters in an acute toxicity test according to the OECD 202 guideline. All experiments revealed 48 h-EC50 values > 100 mg TiO2/L (nominal). 48 h EC10 values < 100 mg/L were only determined for uncoated TiO2 materials in well-spring water (48 h-EC10: 3.7 mg/L) and M4 medium (48 h-EC10: 76.4 mg/L), as well as for the coated TiO2 material T-Lite MAX in M4 medium (48 h-EC10: >85.1 mg/L). Exposure concentrations were not measured.
Experiments, which were performed in M4 medium are not considered to be relevant for hazard assessment purposes of nano-TiO2, because M4 medium contained EDTA.
Executive summary:

During all acute toxicity tests the measured pH of the test dispersions remained within the range of 7.1 and 8.7 and did not vary by more than 1.0 unit in any given test. During the chronic toxicity study the pH remained within the range of 7.8 and 8.4. The temperature ranged from 20.0 to 21.0°C during all acute tests and from 19.3 to 21.3 °C during the chronic toxicity study. The oxygen content of the test dispersions in all acute toxicity tests was above >= 3mg/L.

Endpoint:
short-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: No validation of TiO2 application to test vessel; Test medium: standard (ISO medium) and non-standard medium (diluted ISO medium); No acclimatisation of test organisms to test medium; Tests with simulated solar radiation: non-standardized light conditions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Principles of method if other than guideline:
Tests were performed either in ISO medium or in 10-fold diluted ISO medium. Furthermore, tests were either irradiated with laboratory light or simulated solar radiation.
GLP compliance:
no
Remarks:
Raw data was documented and balances as well as pipettes were used in the style of GLP. The quality assurance unit was not present and did not check the report; the report was not stored in an archive.
Analytical monitoring:
yes
Details on sampling:
SEDIMENTATION EXPERIMENT:
- TiO2 concentration (nominal): 0, and 1.3 mg/L
- Test medium: 10-fold diluted ISO medium
- Time points: 0 h, 24 h, 48 h
- Number of replicates: 3
- Sampling method: The test medium was sampled by gently placing the tip of a glass pipette directly beneath the water column surface
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition of test material into test vessel (dispersion): no
- Sonication of stock (duration): yes, 15 minutes by using ultrasound probe (200 W, pulsed with 0.2 s pulse and 0.8 s pause at 100% power)
- Solvents: no
- Filtration or other separation of test material from test solution: no
- Appearance of solution: no information
- Renewal of test solution: no
- Test medium: deionised water

Stock suspensions (1 g/L, n=8) were prepared by dispersing the TiO2 materials (NM 101 and NM 102; each) in deionised water and sonicating them for 15 min. Working suspensions (100 or 10 mg/L, n = 3) were diluted from stock suspensions with deionised water. For the acute toxicity experiments, different TiO2 concentrations were prepared in diluted (1:10) or undiluted ISO medium (ISO 6341).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna Straus, clone 5
- Source: laboratory culture
- Age at study initiation (mean and range, SD): < 24 h old neonates
- Feeding during test: no


ACCLIMATION
- Acclimation period: not performed
- Culture conditions: One daphnid per test beaker was cultured in 100 ml Elendt M4 medium in a climate chamber (20 °C) with a photoperiod of 16/8 h
- Type and amount of food: Desmodesmus subspicatus (0.2 mg C/daphnid)
- Feeding frequency: 3 times a week
- Medium was renewed once a week
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
no data
Test temperature:
20 ± 1 °C
pH:
6.14-7.84 (for all tests); pH variation in each test was less than 1.5 pH units
Dissolved oxygen:
above 3 mg/L for all tests
Salinity:
no data
Conductivity:
not stated
Nominal and measured concentrations:
In diluted (1:10) ISO medium, the nominal concentrations under laboratory light conditions for NM 102 were 0.6 to 50 mg n-TiO2/L and for NM 101 5 to 100 mg/L.
In ISO medium, the nominal concentration for NM 101 was 12-100 mg/L and for NM 102 0.21 to 50 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: test beaker
- Material, size, headspace, fill volume: filled with 10 mL medium
- Aeration: no information
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

All tests were at least performed twice (n=8) except the test with NM 101 in ISO medium and laboratory light, which was only performed once (n=4).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO water (ISO, 1996) and 10-fold diluted ISO medium

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark
- Light sources:
- Laboratory light (LL): A normal fluorescent tube (Osram Lumilux cool white, HO 49 W/840), which was enclosed by a polycarbonate cover was used for testing under laboratory light.
- Simulated solar radiation (SSR): A metal vapor lamp emitting visible radiation comparable to sunlight (280-1000 nm) was used (Bright Sun UV Desert, 70 W, Lucky Reptile, Waldkirch, Germany) as light source for the testing with SSR.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mobility after 24 and 48 h
Reference substance (positive control):
not specified
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Hombikat UV 100; particle size 10 nm; laboratory light; ISO medium
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
PC 105; particle size 25 nm, laboratory light; ISO medium
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Hombikat UV 100; particle size 10 nm, laboratory light; ISO medium
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
PC 105; particle size 25 nm, laboratory light; ISO medium
Basis for effect:
mobility
Details on results:
- For the nano-sized TiO2 materials NM 101 and NM 102 no significant immobilisation was observed under laboratory light conditions in a standard test medium (ISO medium, according to ISO 6341).
- In non-standardized 10-fold diluted ISO medium no significant immobilisation was observed for the TiO2 material NM 102 under laboratory light conditions, whereas slight effects on mobility of daphnids were observed for NM 101 (48 h-EC50: 79.52 mg/L).

Under co-exposure with simulated solar light, which are non-standardized light conditions, lower EC50 values were detected (please refer to the tables below).
Reported statistics and error estimates:
Data were statistically analysed with ToxRat® Professional (version 2.10, ToxRat solutions GmbH). Concentration response functions were fitted to the data using probit analysis using linear maximum likelihood regression. The median effective concentration (EC50) was calculated from this function. Significant differences to the control (*P<0.05) were determined using Fisher’s Exact Binominal Test with Bonferroni Correction.
Significant differences between the treatment groups of DLS, ELS and sedimentation experiments were determined using student-t test (two sided, *P<0.05).

Table 1: EC50, LOEC, and NOEC values (nominal) derived from the acute toxicity test with the titanium dioxide nanomaterial NM-101 and NM-102 performed with simulated solar radiation (SSR) in ISO medium

TiO2 material
(h of exposure)
EC50
(mg/L)
95%-CL
upper/lower (mg/L)
LOEC
(mg/L)
NOEC
(mg/L)
NM 101 (24 h) n.c. n.c. > 50.0 ≥ 50.0
NM 101 (48 h) 2.9 3.5/2.3 ≤ 1.3 < 1.3
NM 102 (24 h) 16.1 24.3/11.5 1.3 0.5
NM 102 (48 h) 1.1 1.4/0.8 ≤ 0.2 < 2.0

PARTICLE SIZE DISTRIBUTION IN STOCK DISPERSION

The hydrodynamic diameters (HD) and zeta potentials (ZP) of NM 101 account to 488.9 nm and -33.4 mV and those of NM 102 to 621 nm and +12 mV. The polydispersity index (PDI) ranged between 0.26 and 0.33 for the nanomaterials.

PARTICLE SIZE DISTRIBUTION IN TEST MEDIUM

- NM 101: HDs decreased from 752.5 ± 36.8 (PDI: 0.33 ± 0.01) at test start (0 h) to around 250 nm after 48 h. Zeta potential remained at - 20 mV over the entire testing period.

- NM 102: Due to enhanced precipitation, no DLS measurements were possible; Zeta potentials ranged from -12.6 ± 0.95 mV to +7.65 ± 1.52 mV over the testing period.

SEDIMENTATION EXPERIMENT

-Only 10 to 20% of the nominal concentrations of all TiO2 materials was determined in the upper water phase of the test medium at test start.

Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the nano-sized TiO2 materials NM 101 (particle size: 7-10 nm) and NM 102 (particle size: 15-25 nm) did not affect the mobility of Daphnia magna Straus neonates in an immobilisation toxicity test in ISO medium, which was performed according to OECD 202 (2004). The determined 48-h EC50 values were >50 mg n-TiO2/L (nominal) for NM 102 and >100 mg n-TiO2/L (nominal) for NM 101, respectively, under laboratory light conditions.

Tests performed in non-standardized test medium (10-fold diluted ISO medium), revealed that NM 102 did not affect the mobility of Daphnia magna up to the highest tested concentration of 50 mg/L. However, NM 101 in 10-fold diluted ISO medium affected the mobility of Daphnia magna, resulting in a 48-h EC50 value of 79.52 mg n-TiO2/L (nominal). It remains unclear, whether effects of NM 101 on the mobility of Daphnia magna at these high concentrations were caused by intrinsic properties of NM 101, or whether swimming was impaired by physical impairment. Thus, these results cannot be used for the environmental hazard assessment of nano-sized TiO2.

Toxicity of NM 101 and NM 102 was more pronounced under non-standardized light conditions (solar radiation) than under laboratory light conditions.
Endpoint:
short-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: No quantification of titanium concentration of test dispersion; Effects observed after 72 hours cannot be considered reliable as the organisms were not fed during testing, which might have increased their sensitivity.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Longer exposure duration: 72 h
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition of test material into test vessel (dispersion): no
- Sonication of dispersions: A stock dispersion of 1.0 g/L nTiO2 was prepared by dispersing the nanoparticles in ultrapure water (Millipore, Billerica, MA, USA) with sonication for 10 min (50W/L at 40 kHz); a further 10 min of sonication was conducted immediately before dosing each day. Test dispersions of nTiO2 were prepared immediately prior to use by diluting the stock dispersion with daphnia culture medium.
- Solvents: no
- Filtration or other separation of test material form test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: yes, daily
- Test medium: ISO test water according to ISO 7346-3 (1996)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: Carolina Biological Supply Company (USA)
- Age at study initiation (mean and range, SD): 6 - 24 h old
- Feeding during test: daphnids were not fed during the test

ACCLIMATION
- Acclimation conditions (not the same as test): The culture medium was renewed two times each week, and the daphnids were fed daily with food purchased purchased from the same company. The culture was maintained at a constant temperature (22 ± 2 °C) with a natural light–dark cycle.
- Type and amount of food: no further data
- Feeding frequency: daily
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
22 ± 2 °C
pH:
no data
Dissolved oxygen:
no data
Salinity:
not required
Conductivity:
--
Nominal and measured concentrations:
nominal concentration: 0, 0.1, 0.5, 1, 5, 10, 50, and 100 mg n-TiO2/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Material, size, headspace, fill volume: glass, 50 mL, 30 mL test solution
- Renewal rate of test solution (frequency): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1

TEST MEDIUM PARAMETERS
The daphnia culture medium was prepared according to OECD 202 (2004) and ISO 7346-3 (1996) consisting of 64.75 mg/L NaHCO3, 5.75 mg/L KCl, 123.25 mg/L MgSO*7H2O, and 294 mg/L CaCl2*2H2O.

OTHER TEST CONDITIONS
- Photoperiod: natural light/dark rhythm
Reference substance (positive control):
yes
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
mobility
Remarks on result:
other: 95 % CL: 0.87 - 2.45; Results are not reliable as the organisms were not fed during the testing period.
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
2.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
mortality
Remarks on result:
other: 95 % CL: 1.22 - 2.86; Results are not reliable as the organisms were not fed during the testing period.
Reported statistics and error estimates:
Endpoint estimation:
The 48 and 72 h EC50 (immobilization), and LC50 (mortality) values as well as their associated 95% confidence intervals (95% CI) were calculated using a probit method (US EPA Probit Analysis Program, Ver. 1.5, http://www.epa.gov/nerleerd/stat2/probit.zip).

Statistical analysis:
All experiments were repeated three times independently, and data were recorded as the mean with standard deviation (SD). Linear regression was used to determine Michaelis–Menten constants. For toxicity tests, a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons was used to detect significant differences between the control and treated groups. In all data analyses, a p-value < 0.05 was considered statistically significant.

Characterisation of nanomaterials according to Zhang et al. (2997):
For the TiO2 concentrations, the mean values were calculated from the three replicates and expressed with standard deviation (n = 3). The homogenicity of variance was checked out and a one-way analysis of variance (ANOVA) was then performed to assess the significance of differences observed TiO2 nanoparticles. All statistical analyses were conducted at a significance level of 0.05.
Validity criteria fulfilled:
not specified
Remarks:
Control mortality after 48 h < 10%; Oxygen content not reported
Conclusions:
Daphnia magna (age, < 24 h) exposed to dispersion of the nano sized TiO2 material P25 (21 nm) in an acute toxicity assay according to OECD 202 did not show immobilisation after a standard exposure period of 48 h (48 h EC50 of > 100 mg n-TiO2/L, nominal). Effects observed after 72 hours cannot be considered reliable as the organisms were not fed during testing, which might have increased their sensitivity.
Endpoint:
short-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: no quantification of titanium concentration of test dispersions; Tests in artificial freshwater were performed in M4 medium, containing EDTA
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): no
Sonication of stock dispersion: 2 acute toxicity tests were run with non nano-scale TiO2. For both tests, a stock dispersion of 100 mg/L was prepared. The dispersions were ultrasonicated for 5 min after transferring the test item into either artifical Elendt M4 medium (M4) or well spring water (SW).
Solvents: no
Filtration or other separation of test material from test dispersion: no
Appearance of dispersion: In all tests, except those using filtered test medium, the test compounds were visually observed to not completely disperse at concentrations levels 10-100 mg/L. The dispersions appeared turbid and settled material was observed at the bottom of the test vessels after 24 h and 48 h. During the test period further sedimentation of particles was visually observed.
Renewal of test dispersion: no
Test medium: Either artificial Elendt M4 medium (M4) or well-spring water of the River Selz in south-western Germany (SW)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: D. magna Strauss
- Source: laboratory stock culture at the test facility
- Age at study initiation (mean and range, SD): <=24 h old
- Food type: suspension of the green algae Desmodesmus subspicatus
- Frequency: daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2.02 - 2.86 mmol/L M4
5.95 mmol/L SW
Test temperature:
20±1 °C
pH:
7.1 - 8.7
Dissolved oxygen:
> 3 mg/L
Salinity:
not relevant
Nominal and measured concentrations:
nominal concentrations: 1, 10, and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass tubes
- Material, size, headspace, fill volume: 10 mL
- Renewal rate of test solution (frequency/flow rate): static
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 1

- Test concentration: max. 5 (from 0.01-100 mg/L); factor: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 (according to OECD 202)
- Source/preparation of dilution water: SW river selz, southwest Germany
- Total organic carbon: 1.3 mg/L SW
- Conductivity: 577 - 605 µS/cm M4
- Conductivity: 1108 µS/cm SW

Experiments which were performed in M4 medium are not considered to be relevant for hazard assessment purposes of nano-TiO2, because the test medium contained EDTA.

Oxygen content and pH of the test media were measured at the beginning and at the end of the test in one replicate per control and per test concentration level (Multiline P4, WTW, Weilheim, Germany).

OTHER TEST CONDITIONS
- Photoperiod: 16h light/ 8h dark
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks:
in SW medium
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
13.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks:
in SW medium
Details on results:
Test in M4 medium:
48 h-EC50 (mobility): > 100 mg/L
48 h-EC10 (mobility): 91.2 mg/L
Results with reference substance (positive control):
Reference acute toxicity tests using potassium dichromate were performed regularly at intervals of 1–2 months, whereby the median effective concentration (EC50) after 24 h ranged from 0.6 to 1.4 mg/L during the last 10 months, thus meeting the validity criterion according to the OECD guideline.
Reported statistics and error estimates:
Effective concentrations (EC10 and EC50 values) for the parameter immobilization were calculated with the help of the results obtained from the 48-h acute toxicity tests. The dose–response curve was fitted via the probit model according to Finney (1971). If the goodness-of-fit test Pearson Chi-Square was significant (p 6 0.05), high or low doses were excluded from the evaluation. This analysis was only performed, if interpolation was possible. In those cases were extrapolation was necessary the EC10 or EC50 were given as >100 mg/L. All calculations were done withthe help of Microsoft Excel 2000.

During all acute toxicity tests the measured pH of the test dispersions remained within the range of 7.1 and 8.7 and did not vary by more than 1.0 unit in any given test. During the chronic toxicity study the pH remained within the range of 7.8 and 8.4. The temperature ranged from 20.0 to 21.0°C during all acute tests and from 19.3 to 21.3 °C during the chronic toxicity study. The oxygen content of the test dispersions in all acute toxicity tests was above >= 3mg/L.

Validity criteria fulfilled:
yes
Remarks:
according to OECD guideline
Conclusions:
Daphnia magna (< 24 h old) were exposed to dispersions of a pigment grade TiO2 material either in artificial (M4 medium) or natural water in an acute toxicity test according to the OECD 202 guideline. Determined 48 h-EC50 values were > 100 mg TiO2/L (nominal) in both media, and 48 h-EC10 values were 13.8 mg/L (nominal) and 91.2 mg/L (nominal) in either natural or artifical water. Exposure concentrations were not measured. Results of experiments, which were performed in M4 medium are not considered to be relevant for hazard assessment purposes of nano-TiO2, because M4 medium contained EDTA.

Description of key information

 Dispersions of microsized TiO2 are not acutely toxic to freshwater and marine invertebrates up to at least 1000 and 10,0000 mg/L (EC/LC50: > 1000 and > 10,000), respectively. Considering the low solubility of microsized TiO2, it is further concluded that microsized TiO2 is not acutely toxic up to its solubility limit. Dispersions of nanosized TiO2 are also not acutely toxic to freshwater and marine invertebrates up to at least 100 mg TiO2/L (EC50: > 100 mg/L).

Key value for chemical safety assessment

Additional information

Microsized TiO2:

Dispersions of four different microsized TiO2 materials did not affect the mobility of Daphnia magna in an acute toxicity test according to US EPA (660/8-87/011; 1987) and ASTM (Standard E729, 1986). Unbounded 48 h EC50 values of > 1000 mg TiO2/L (nominal) were derived for all four materials (Haley and Kurnas, 1993). Johnson et al. (1986) observed that dispersions of the microsized TiO2 material Titanox 1000 (particle size: 5-6 µm) did not affect the mobility of first instar Daphnia magna in an acute toxicity test according to OECD 202 and US EPA (660/8-87/011; 1987). The determined unbounded 48 h EC50 value was > 1000 mg TiO2/L (nominal). Wiench et al. (2009) also did not observe acute toxicity of dispersions of microsized TiO2 (200-300 nm) up to 100 mg/L in artificial or natural water in acute toxicity tests with Daphnia magna according to OECD 202. However, the artificial water contained EDTA so that the respective EC50 may not be considered conservative. One supporting acute toxicity test with microsized TiO2 material (Tiona AT 1, 200 nm) confirms that microsized TiO2 is not acutely toxic to Daphnia magna since the 48 h EC50 was > 50 mg/L. However, the test was performed in diluted ISO medium that does not meet the requirements of OECD 202 and results of this study are considered supportive. 

Microsized TiO2 up to at least 10,000 mg/L was also not acutely toxic to marine invertebrates: Thomson et al. (2007) exposed the marine crustacean Acartia tonsa (17-25 d old)in an acute toxicity test according to ISO 14669 (1999) to the supernatant of a microsized TiO2 dispersion, which was prepared by stirring the microsized TiO2 dispersion for 24 h and letting it settle for 1 h. Results show that the tested material did not affect the survival of Acartia tonsa, and an unbounded 48 h LC50 value of > 10,000 mg/L (nominal) was reported.

Finally, it can be concluded that dispersed microsized TiO2 is not acutely toxic to freshwater and marine invertebrates up to concentrations of at least 100 (EC50) and 10,000 mg/L (LC50), respectively.

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). Thus, all acute invertebrate toxicity tests with microsized TiO2 were performed at TiO2 water concentrations above the solubility limit of microsized TiO2. It is further concluded that microsized TiO2 is not acutely toxic to freshwater invertebratesup to its solubility limit.

Nanosized TiO2:

Several studies investigated the acute toxicity of nanosized TiO2 dispersions to different freshwater invertebrates, including Daphnia magna, Daphnia similis, Daphnia pulex, Ceriodaphnia dubia and Hyalella azteca and results indicate that nano-TiO2 is not acutely toxic to these organisms up to at least 100 mg/L (EC/LC50: > 100 mg/L).

The nanosized TiO2 material P25 was tested for its acute toxicity towards daphnids by exposing either Daphnia magna (Amiano et al. 2012, Zhu et al. 2010), Ceridaphnia dubia (Griffit et al. 2008), Daphnia pulex (Griffit et al. 2008), or Daphnia similis (Clemente et al. 2014) for 48 h to dispersed P25 in standard acute toxicity tests according to OECD 202 or ASTM guidelines. Griffit et al. (2008) determined an unbounded 48 h EC50 value of > 10 mg/L (nominal) for P25 after exposing C. dubia and D. pulex under semi-static test conditions (ASTM standard guideline). Under laboratory light conditions, Clemente et al. (2014) derived an unbounded 48 h EC50 value of > 1000 mg/L (nominal) for D. similis (OECD 202). Zhu et al. (2010) determined an unbounded 48 h EC50 value of > 100 mg/L (nominal) for D. magna (OECD 202). Zhu et al. (2010) extended the exposure duration to 72 h and observed toxicity of P25 to D. magna but the 72 h results are not reliable since the organisms were not fed during the test and may have become more sensitive.

However, Amiano et al. (2012) observed 48 h EC50 values of 19.3 mg/L (measured) and 27.8 mg/L (measured) for D. magna exposed to dispersed P25 in ISO medium or river water under dark conditions (OECD 202). According to Amiano et al. (2012) “P25 was highly attached to the daphnids’ appendages, limiting their mobility” and being “a physical impediment to swimming” and possibly particle attachment resulted in a “blockage of gills” at TiO2 concentrations between 10-100 mg/L. Thus, the EC50 derived by Amiano et al (2014) may not be a “true EC50” since this threshold is not a reliable estimate of the intrinsic toxicity of titanium dioxide, and results of this study cannot be considered relevant for the purposes of hazard classification.

Furthermore, two studies were performed with dispersions of five additional nanosized TiO2 materials and confirm that nano-TiO2 is not toxic to aquatic freshwater invertebrates. Wiench et al. (2009) exposed Daphnia magna (< 24 h old) to dispersions of four nano-TiO2 materials differing in crystalline phase, size, and coating and dispersions were prepared with different techniques in artificial M4 medium or natural waters (OECD 202). Unbounded 48 h EC50 values of > 100 mg TiO2/L (nominal) were derived in all experiments. However, the M4 medium contained EDTA so that the respective EC50 may not be considered conservative. Wyrwoll et al. (2016) investigated if dispersions of the nanosized TiO2 materials NM 101 (particle size: 7-10 nm) and NM 102 (particle size: 15-25 nm) affected the mobility of Daphnia magna in ISO medium (OECD 202). The determined unbounded 48 h EC50 values were > 50 mg n-TiO2/L (nominal) for NM 102 and > 100 mg n-TiO2/L (nominal) for NM 101, under laboratory light conditions. Wyrwoll et al. (2016) tested NM 101 and NM 102 also in diluted ISO medium and observed a comparable toxicity or lack thereof (i.e. 48 h EC50: > 50 mg/L NM 102 and 48 h EC50: 79.52 mg/L NM 101) of both n-TiO2 materials compared to tests performed in undiluted ISO medium. However, the diluted ISO medium is not a standardized test medium and it does not meet the hardness recommendation of OECD 202. Thus, effect values of tests in diluted ISO medium are considered supportive. Two supporting studies on the acute toxicity of nanosized TiO2 to Daphnia magna indicate that nanosized TiO2 is not acutely toxic to Daphnia magna up to at least 100 mg/L (Ma et al. 2012, Cupi et al. 2016). However, inappropriate life stages were tested (Ma et al. 2012) or tests were performed in M7 medium containing EDTA and organisms were not acclimated to the very soft medium (Cupi et al. 2016). In sum, it is concluded that nanosized TiO2 is not acutely toxic to daphnids up to concentrations of 100 mg/L.

Additionally to the daphnia studies, the epibenthic freshwater organism Hyalella azteca was exposed via the water phase to dispersed nanosized TiO2 material P25 (25.1 ± 8.2 nm) in a test system containing silica sand as sediment (Li et al. 2014). Results indicate a low toxicity to juvenile Hyalella azteca (age, 7-8 d) under laboratory light conditions resulting in a 96 h LC50 of 631 mg/L (nominal). Measurements of TiO2 concentrations in the water phase point to a sedimentation of more than 77% of the nominal TiO2 so that these benthic organisms were exposed to concentrations at the bottom far exceeding the water TiO2 concentrations. Furthermore, it may be assumed that particles attach to the organisms at concentrations of dispensed nanosized TiO2 above 100 mg/L resulting possibly in a blockage of the gills of H. azteca. Thus, the EC50 derived by Li et al (2014) may not be a “true EC50” since this threshold is not a reliable estimate of the intrinsic toxicity of titanium dioxide, and results of this study cannot be considered relevant for the purposes of hazard classification.

Nano-TiO2 is also not acutely toxic to the marine organism Artemia salina up to dispersed TiO2 concentrations of at least 100 mg/L (EC/LC50). The acute toxicity of nanosized TiO2 dispersions (10-30 nm) to Artemia salina (nauplii and adults) was investigated after 24 h and 96 h according to OECD 202 (Ates et al. 2013). Significant mortality or toxicity was not observed at any dose resulting in an unbounded 96 h LC50 of > 100 mg/L (nominal). To the contrary, Clemente et al. (2013) found that dispersions of the nano sized TiO2 material Aeroxide P25 (particle size: 25 nm) affected the mobility of Artemia salina nauplii in an acute immobilisation test (similar to OECD 202). The determined 48 h EC50 amounts to 284.81 mg TiO2 NP/L (nominal) under laboratory light conditions. It may be assumed that particles attach to the organisms at concentrations of dispersed nanosized TiO2 above 100 mg/L resulting possibly in a blockage of the gills of A. salina. Thus, the EC50 derived by Ates et al (2013) may not be a “true EC50” since this threshold is not a reliable estimate of the intrinsic toxicity of titanium dioxide, and results of this study cannot be considered relevant for the purposes of hazard classification. Furthermore, in a supporting study of Minetto et al. (2017) a significant effect of titanium dioxide nanoparticles (P25, particle size: 15-60 nm) on the mortality of Artemia franciscana nauplii was observed after 48 h exposure in ASTM artificial seawater, and a 48 h LC50 of 18 mg TiO2 NP/L (nominal; scenario A & C) was determined. Since test organisms were not acclimatised to the test medium it is expected that sensitivity of test organisms might have increased. Furthermore, exposure concentrations were not verified. Thus, results are only considered supporting.

Several studies examined the influence of solar radiation on the toxicity of nanosized TiO2 to invertebrates including freshwater species such as Daphnia magna (Amiano et al. 2012, Wyrwoll et al. 2014), Daphnia similis (Clemente et al. 2014) and marine species such as Artemia salina (Clemente et al. 2014) and observed a pronounced toxicity compared to the low toxicity or lack thereof observed in tests at standard light conditions. However, these phototoxicity experiments were performed under non-standard light conditions and respective results cannot be considered relevant for the purposes of hazard classification. 

Based on a weight of evidence approach, it is concluded that dispersed microsized and nanosized TiO2 are not acutely toxic to invertebrates up to at least 100 mg TiO2/L (EC/LC50: > 100 mg/L) in freshwater and in marine water. Considering the low solubility of microsized TiO2, it is further concluded that microsized TiO2 is not acutely toxic up to its solubility limit.