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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
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): yes
Stirring of stock solution (duration): 2 min (vortex)
Stirring of test solution: aeration assisted in maintaining homogeneity but aggregation could not be avoided
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: Water visibility decreased substantially with increasing concentration of TiO2 NPs
Test organisms (species):
Carassius auratus
Details on test organisms:
TEST ORGANISM
- Common name: goldfish
- Source: local pet shop
- Body length at study initiation: 5.5 +-0.7 cm
- Body weight at study initiation: 4.53 +- 0.06 g


ACCLIMATION
- Feeding frequency: daily
- Food: TetraFin Goldfish flakes, Germany
- Amount of food: 0.5% of mean body weight of the fish
- cultures were maintained at 23 +- 2°C and pH of 6.8 enriched with oxygen
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
23 +- 2°C
pH:
6.03-6.63
Dissolved oxygen:
6 ppm +-1
Nominal and measured concentrations:
10 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 10L aquarium
- Renewal rate of test solution: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
TiO2 as dispersion
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
TiO2 as dispersion
Basis for effect:
mortality
Details on results:
The growth of the fish was inhibited with increasing exposure dose of nTiO2. While the controls showed about 8.1% increase in body weight, those exposed to 10 mg/L showed a small increase (1.8%) in body weight, and those exposed to 100 mg/L showed about 19.7% decrease in body weight at the end of the experiments.
Validity criteria fulfilled:
yes
Conclusions:
Nano TiO2 did not affect mortalityof Carassius auratus at the highest test concentration (100 mg nTiO2/L as dispersion) in an acute toxicity test (OECD 203).
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sub-chronic study; well performed and well documented with minor deviations
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)
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): yes, via stock solution
Stirring of stock solution (duration): overnight
Subsequent sonication of stock solution (duration): 1 h and each day before addition to tanks
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: no information

Stock dispersions (500 ml) of 10 g/L nano Ti02 (nominal concentrations) were prepared in ultrapure water and stored in acid-washed Nalgene bottles.Stocks were stirred overnight (2400 rpm) and subsequently sonicated (4°C, 35 kHz frequency) for 1 h. The sonication step was repeated each day immediately prior to dosing the experimental tanks to ensure dispersed material was added to the tanks.

Aerated dechlorinated Plymouth tap water. Electrolyte composition: 0.75 ± 0.11 mmol/l Na+, 0.06 ± 0.01 mmol/l K+, 0.52 ± 0.07 mmol/l Ca+2 and 0.06 ±0.01 mmol/l Mg+2, NH3 0.08 ± 0.003 mg/L
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Juvenile rainbow trout were obtained from Torre fishery, Watchet, Somerset, UK
Fish weighing 24.0±3.7 g (n=32)
Feeding: twice daily with EWOS, Westfield, UK until 48h prior to experimentation
Titanium concentration in water: below the detection limit
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
15 ± 1 °C
pH:
7.2 ± 0.2
Dissolved oxygen:
8.3 ± 0.5 mg/l
Salinity:

Electrolyte composition: 0.75 ± 0.11 mmol/l Na+, 0.06 ± 0.01 mmol/l K+, 0.52 ± 0.07 mmol/l Ca+2 and 0.06 ±0.01 mmol/l Mg+2, NH3 0.08 ± 0.003 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 1.0 mg/L TiO2.
Measured concentrations of titanium metal in water samples taken 5 min after dosing were <1.3 µg/L (control), 0.64 ± 0.01 mg/L, corresponding to 1.07 ± 0.01 mg/L TiO2 in the treatments.
Details on test conditions:
TEST SYSTEM
- Test vessel: 20L glass tanks
- Renewal rate of test solution: 12h each
- No. of organisms per vessel: 14
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 14:10 h
- Light intensity: laboratory light (350-400 lux lightning)

TEST CONCENTRATIONS
- 2 concentrations (0 (control), 1 mg/L TiO2)

Each tank was dosed with 2 mL of the appropriate stock solution (10g/L, stirring overnight and sonicated after) with the aeration rapidly spreading the material in the tank water. An 80% water change, re-dosed with 1.6 mL of TiO2. Fishes were fed twice (day 8 and 12) with a 1.5% body weight maintenance ration. This was done post water change but prior to re-dosing to minimise the risk of ingestion of TiO2 during feeding.
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25 as dispersion
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25 as dispersion
Basis for effect:
mortality
Details on results:
There was no overall material-type effect on animal behaviour.
Validity criteria fulfilled:
not specified
Conclusions:
No significant mortality of rainbow trout was observed at the single test concentration of nano TiO2 (1 mg TiO2/L as dispersion) in a study similar to OECD 204.
Endpoint:
short-term toxicity to fish
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; not all validity criteria assessable: dissolved oxygen concentration not measured during the test period. However, control mortality meets validity criteria and hatching rate not affected.
Qualifier:
equivalent or similar to
Guideline:
other: OECD 236: Fish Embryo Test, FET
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): yes, dilution of stock solution
Sonication of stock solution (duration): 10 min
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution:

- Method: The stock suspension of 1 g n-TiO2/L was prepared in Milli-Q water by sonication for 10 min using a high frequency probe operated at an amplitude of 20% (120 W/L). Immediately after sonication, aliquots were removed for the preparation of suspensions containing 1, 10, 100 mg/L of n-TiO2 under the same conditions used for the bioassays (with dilution in the embryo exposure medium).
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Age at study initiation (mean and range, SD): embryos (1-h post fertilization)
- Feeding during test: no data
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
5°dGH
Test temperature:
26.0 ± 1 °C
pH:
7.5 ± 0.5
Dissolved oxygen:
6 ± 0.6 mg/L
Salinity:
not relevant
Nominal and measured concentrations:
nominal concentration: 0, 1, 10, and 100 mg/L n-TiO2
Details on test conditions:
TEST SYSTEM
- Test vessel: 24-well polystyrene plare
- Material, size, headspace, fill volume: 2 mL suspension volume
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Conductivity: 600 ±10 µS/cm
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25 as dispersion
Basis for effect:
mortality
Remarks:
(embryo, 1h pf, survival & hatching rate)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25 as dispersion
Basis for effect:
mortality
Remarks:
(embryo, 1h pf, survival & hatching rate)
Details on results:
Additionally to the acute toxicity, eclosion rate, total larva length, and specific activities of catalase (CAT), glutathione S-transferase (GST) and acid phosphatase were determined without any significant effects. However, nano­Ti02 adhered to the chorion, forming an external white layer that hindered a clear view of the embryo, and it was not possible to properly evaluate the occurrence of malformations during the embryonic period. Further, 5% mortality of the larvae exposed to 100 mg/L TiO2 P25 A under UV light were observed.

Please note that only results of tests performed with TiO2 nanopowder AEROXIDE TiO2 P 25 are summarised (see above).

Table 1: Analysis- DLS measurements of the 100 mg/L suspension of the nano TiO2 in the embryo exposure medium (Results are presented as the mean (± standard deviation) of 3 readings.

measurment oft he 100 mg/L suspension (nano-TiO2)

0 h

3 h

6 h

24 h

Z-average1(nm)

1224.6 (± 166.1)

1903.3 (± 12.6)

2132.0 (± 259.9)

4075.6 (± 1476.5)

PdI2

0.2 (± 0.02)

0.4 (± 0.07)

0.8 (± 0.1)

1.0 (± 0.0)

Peak3(nm)

1252.0 (± 116.2)

1109.3 (± 95.7)

662.0 (± 233.2)

470.8 (±403.8)

PZ4(mV)

-13.2 (± 0.1)

-15.2 (± 0.6)

-19.0 (± 0.9)

-21 (± 2.9)

1) Z-average: Average size of the particle in suspension; 2) PdI: Polydispersion index; 3) Peak: Size of the main particle population; 4) PZ: zeta potential

Validity criteria fulfilled:
yes
Conclusions:
The 96-h survival of Danio rerio embryos was not affected in an acute fish embryo toxicity test (OECD TG 236) at 100 mg n-TiO2/L (as dispersion).
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sub-chronic study; well performed and well documented with minor deviations
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)
Principles of method if other than guideline:
Fish were exposed to different concentration of the test materials for 14 days. Mortality and several biochemical endpoints (haematology, blood plasma, analysis of the gill, liver, skinned muscle and brains) were measured after 0, 7 and 14 days.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): yes
Sonication of stock solution (duration): 6 h plus 30 min directly before dosing
Stirring of test solution: aeration and water flow in the tank dispersed each dose around the tank within seconds in all experiments
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: Dispersion was confirmed by transmission electron microscopy

Volumes of TiO2 NP stock were adjusted on re-dosing to reflect the 80% water change every 12 h.

Test medium: dechlorinated Plymouth tap water.
Electrolyte composition: 0.5 mmol/l Na+, 0.1 mmol/l K+, 0.4 mmol/l Ca+2 and 0.1 mmol/l Mg+2.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Juvenile rainbow trout were obtained from Hatchlands Trout Farm, Rattery, Devon.
Fish weighing 28.1±0.4 g.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Hardness:
50 mg CaCO3/l
Test temperature:
14±1 °C
pH:
7.11±0.01
Dissolved oxygen:
87.0 +/-0.25%
Nominal and measured concentrations:
Nominal: 0 (control), 0.1, 0.5 and 1.0 mg/L TiO2.
Measured TiO2 concentrations after dosing the tanks were 95, 98 and 97% of the nominal target values for the 0.1, 0.5 and 1.0 mg TiO2/L treatments, respectively. Before the 12h water change, measured concentrations were 89, 85 and 86% of the nominal values for the 0.1, 0.5 and 1.0 mg TiO2/L treatments, respectively.
Details on test conditions:
Semi-static regime: water change every 12 h with re-dosing after each change.
Fish were not fed 24 h prior to, or during the experiment. Except for on day 10 after the water change and prior to re-dosing with TiO2.
Photoperiod was 12 h light and 12 h dark.
Fish were randomly sampled on days 0 (initial fish from the stock), 7 and 14 for haematology, plasma ions, tissue electrolytes, histopathology and biochemistry.
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25 as dispersion
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25 as dispersion
Basis for effect:
mortality
Details on results:
Results for biochemical endpoints (data not relevant for mortality):

Haemoglobin, haematocrit, red blood cells, white blood cells, mean erythrocryte haemoglobin content, mean erythrocyte volume, plasma K+:

14 d NOEC >= 1 mg/L nominal test mat.
14 d EC50 > 1 mg/L nominal test mat.

other: Plasma Na+:

14 d NOEC 0.5 mg/L meas. (initial) test mat.
14 d LOEC 1 mg/L meas. (initial) test mat.

Exposure to TiO2 NPs caused some gill pathologies including oedema and thickening of the lamellae. No major haematological or blood disturbances were observed in terms of red and white blood cell counts, haematocrit values, whole blood haemoglobin, and plasma Na+ or K+ concentrations. Tissue metal levels (Na+, K+, Ca2+ and Mn) were generally unaffected but some exposure concentration-dependent changes in tissue Cu and Zn levels were observed, especially in the brain. Exposure to TiO2 NPs caused statistically significant decreases in Na+K+-ATPase activity in gills and the intestine. Thiobarbituric acid reactive substances showed exposure concentration-dependent and statistically significant increases in the gill, intestine and brain, but not the liver during exposure to TiO2 NPs compared to controls. TiO2 NP exposure caused statistically significant increases in the total glutathione levels in the gills, but depletion of hepatic glutathione compared to controls. Total glutathione levels in the brain and intestine were unaffected. Liver cells exposed to TiO2 NPs showed minor fatty change and lipidosis, and some hepatocytes showed condensed nuclear apoptotic bodies. Titanium dioxide nanoparticles are not a major ionoregulatory toxicant, or haemolytic, at the concentration and exposure times used.

Validity criteria fulfilled:
not specified
Conclusions:
No significant mortality of juvenile rainbow trout was observed in a prolonged fish toxicity study at the highest test concentration (1 mg TiO2/L as dispersion).
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sub-chronic study; well performed and well documented with minor deviations
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)
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): via stock solution
Sonication of stock solution (duration): 6 h
Stirring of test solution (duration): yes in tanks during test
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: no information

A 10 g/L stock suspension of TiO2 NPs was made (no solvents) by dispersing the NPs in ultrapure water by sonication (35 kHz frequency) for 6 h. Dosing was done after fish finished feeding to minimise ingestion of Ti02. After each water change, tanks were re-dosed with 80% of the nominated concentrations to bring the final tank concentration back up to the nominal dose (e.g. after an 80 % water change of the 1 mg/L tanks 20 % of the dose remained in the water so 1.6 ml of the 10 g/L stock was added to bring the total concentration back to 1 mg/L). After addition, water was stirred using a glass rod to ensure even mixing. Control tanks were also stirred with glass rods to standardise stress across all tanks.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Zebrafishs (n=360) were obtained from J&K Aquatics Ltd. in Taunton, Somerset, UK, Watchet, Somerset, UK
Fish were held in re-circulating, filtered, dechlorinated, Plymouth freshwater
Fish weighing 0.6±0.01 g (n=300)
Feeding: twice daily with brine shrimp
Acclimatisation period: 14d
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Test temperature:
25.8 ± 0.08 °C
pH:
7.45 ± 0.01
Dissolved oxygen:
7.92 ± 0.01 mg/l (>95% saturation)
Salinity:
ammonium, nitrite, nitrate
0.08±0.01, 0.17±0.02, <22 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 0.1 and 1 mg/L TiO2 nano
Measured concentrations of titanium dioxide inn tanks were 0.04 mg/L (control), 0.12 ± 0.01 mg/L TiO2 and 1.07 ± 0.03 mg/L TiO2 in the treatments.
Details on test conditions:
TEST SYSTEM
- Test vessel: 20L glass tanks
- Renewal rate of test solution: 24h each with 80% water change
- Fish were fed with a single feed of brine shrimp prior to re-dosing the tanks with the appropriate TiO2 each day
- No. of organisms per vessel: 25
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

OTHER TEST CONDITIONS
- Photoperiod: 12:12 h
- Light intensity: laboratory light

TEST CONCENTRATIONS
- 3 respecitvely concentrations (0 (control), 0.1 and 1 mg/L nano TiO2)

An 80% water change, re-dosed with 1.6 mL of TiO2.
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.07 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25 as dispersion
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1.07 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
AEROXIDE TIO2 P25 as dispersion
Basis for effect:
mortality
Validity criteria fulfilled:
not specified
Conclusions:
No significant mortality of Danio rerio was observed at the highest test concentration of TiO2-NP (1.07 mg TiO2-NP/L as dispersion) in a prolonged toxicity test similar to OECD TG 204.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 16-20 2006
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: according to OECD 203, well documented, no quantification of titanium concentration of test solutions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
10-fold increase in concentrations, 5 fish per concentration tested
GLP compliance:
not specified
Analytical monitoring:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): yes
Stirring of test solution (duration): 1 h
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance:
Controls and 0.1 mg/l test concentrations: clear and colorless with no precipitate at test start.
1 mg/l test concentrations: cloudy, with a slight amount of suspended substance present at the start.
10 and 100 mg/l test concentrations: cloudy (white in color) with suspended substance present at the start.

Test solutions were prepared by direct addition of the test material to Haskell Laboratory well water (HLWW) (15 l) and stirring for approximately 60 minutes. HLWW is aerated, passed through a green sand filter to remove iron, and filtered through 50-, 10-, and 0.45 µm filters to remove particulates. The water is heated or chilled as appropriate and distributed through aged PVC-piping.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Rainbow trout (Oncorhynchus mykiss) were purchased from Thomas Fish Company, Anderson, California.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
100-140 mg CaCO3/l
Test temperature:
12.2°C (range 12.1-12.5°C)
pH:
7.4-7.9
Dissolved oxygen:
9.2-10.8 mg/l
Nominal and measured concentrations:
Nominal: 0 (control), 0.1, 1, 10 and 100 mg/l.
Details on test conditions:
Test chambers were stainless steel aquaria (30*30*30 cm) which held approximately 15 l of test solution (test solution depth: 17.5 cm). 5 fish in each test chamber. Each chamber was covered with a glass plate to prevent the fish from escaping.
16 hours light / 8 hours darkness.
Test solutions were not aerated during the test.
Mortality and behavioral observations were made every 24 hours and at test end. Criteria for death were the absence of opercular movement and lack of reaction to gentle prodding.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
n-TiO2 as dispersion
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
n-TiO2 as dispersion
Basis for effect:
mortality
Validity criteria fulfilled:
yes
Conclusions:
Less than 50% mortality of rainbow trout was observed at the highest test concentration (100 mg TiO2/L as dispersion) in an acute toxicity test equivalent to OECD Guideline 203.
Endpoint:
short-term toxicity to fish
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; not all validity criteria assessable: dissolved oxygen concentration not assessable. However, control mortality meets validity criteria and hatching rate not affected, exposure duration 72 h
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 236 (Fish Embryo Acute Toxicity (FET) Test
Deviations:
yes
Remarks:
Exposure duration was only 72 h, instead of 96 h.
Principles of method if other than guideline:
For the main test a static exposure was used to simulate a more realistic exposure scenario. Cell stage of embryos at test initiation was 8-64 instead of 8-16. Exposure duration was only 72 h, instead of 96 h.
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:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): no
Sonication of stock solution (duration): yes, duration not reported
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: no information

Test medium consists of 90 % higher concentrated reconstituted water (HCRW, ISO 1996) and 10 % deionized water or TiO2 suspension.

Stock suspensions (1 g n-TiO2/L) were prepared by suspending the nanomaterial in deionized water using ultrasonication with a microtip (200 W, 0.2 s pulse and 0.8 s pause). Working suspensions were prepared by diluting the stock suspension with deionized water to 100 mg/L. Suspensions were characterized by means of dynamic light scattering. Furthermore, the zeta potentials of the particles in the suspensions were measured with a zetasizer (Malvern Instruments, Worcestershire, UK).
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: Applied Ecology of the Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany
- Age at study initiation (mean and range, SD): fertilized eggs within 8 cell ton 64 cell stage
- photo period: 14 h light: 10 h dark
- temperature: 26 °C
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
24 h
Hardness:
no data
Test temperature:
26 °C
pH:
no data
Dissolved oxygen:
no data
Nominal and measured concentrations:
nominal concentrations: 1, 10, and 100 mg n-TiO2/ L
Details on test conditions:
TEST SYSTEM
- Test vessel: 6-well plates
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: plastic, 10 mL
- Aeration: 6-well plated were covered with an air permeable membrane
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 40 negative control embryos
- No. of vessels per control (replicates): 20 positive control embryos

OTHER TEST CONDITIONS
- Photoperiod: dark

Tests were performed twice. Hence, 20 embryos were tested per TiO2 treatment, corresponding to the number of organisms required for a limit test. Organisms in the second experiment were only exposed for 72 h.
Reference substance (positive control):
yes
Remarks:
3,4- dichloroaniline (3.7 mg/L); should induce >30 % mortality after 96 h
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
TiO2 - Hombikat UV 100 as dispersion
Basis for effect:
mortality
Remarks:
(embryo survival, hatching rate)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
TiO2 - P 105 as dispersion
Basis for effect:
mortality
Remarks:
(embryo survival, hatching rate)
Key result
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
TiO2 - Hombikat UV 100 as dispersion
Basis for effect:
mortality
Remarks:
(embryo survival, hatching rate)
Key result
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
TiO2 - PC 105 as dispersion
Basis for effect:
mortality
Remarks:
(embryo survival, hatching rate)
Details on results:
No effects on survival and hatching rate of embryos exposed to TiO2 in the first experiment after an exposure of 96 h were observed.
Reported statistics and error estimates:
As no dose response relationships were observed no EC values were calculated.
Validity criteria fulfilled:
yes
Conclusions:
Two different titanium dioxide nanomaterials did not affect embryo survival and hatching rate of Danio rerio at the highest test concentration of 100 mg/L (as dispersion) in an acute fish embryo toxicity test (OECD TG 236) after 72 h of exposure.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sub-chronic study; well performed and well documented with minor deviations
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)
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): yes, via stock solution
Stirring of stock solution (duration): overnight
Subsequent sonication of stock solution (duration): 1 h and each day before addition to tanks
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: no information

Stock dispersions (500 ml) of 10 g/L bulk Ti02 (nominal concentrations) were prepared in ultrapure water and stored in acid-washed Nalgene bottles.Stocks were stirred overnight (2400 rpm) and subsequently sonicated (4°C, 35 kHz frequency) for 1 h. The sonication step was repeated each day immediately prior to dosing the experimental tanks to ensure dispersed material was added to the tanks.

Aerated dechlorinated Plymouth tap water. Electrolyte composition: 0.75 ± 0.11 mmol/l Na+, 0.06 ± 0.01 mmol/l K+, 0.52 ± 0.07 mmol/l Ca+2 and 0.06 ±0.01 mmol/l Mg+2, NH3 0.08 ± 0.003 mg/L
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Juvenile rainbow trout were obtained from Torre fishery, Watchet, Somerset, UK
Fish weighing 24.0±3.7 g (n=32)
Feeding: twice daily with EWOS, Westfield, UK until 48h prior to experimentation
Titanium concentration in water: below the detection limit
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
15 ± 1 °C
pH:
7.2 ± 0.2
Dissolved oxygen:
8.3 ± 0.5 mg/l
Salinity:
Electrolyte composition: 0.75 ± 0.11 mmol/l Na+, 0.06 ± 0.01 mmol/l K+, 0.52 ± 0.07 mmol/l Ca+2 and 0.06 ±0.01 mmol/l Mg+2, NH3 0.08 ± 0.003 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 1.0 mg/L TiO2.
Measured concentrations of titanium metal in water samples taken 5 min after dosing were <1.3 µg/L (control), 0.67 ± 0.02 mg/L, corresponding to 1.11 ± 0.02 mg/L TiO2 in the treatments.
Details on test conditions:
TEST SYSTEM
- Test vessel: 20L glass tanks
- Renewal rate of test solution: 12h each
- No. of organisms per vessel: 14
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 14:10 h
- Light intensity: laboratory light (350-400 lux lightning)

TEST CONCENTRATIONS
- 2 concentrations (0 (control), 1 mg/L)

Each tank was dosed with 2 mL of the appropriate stock solution (10g/L, stirring overnight and sonicated after) with the aeration rapidly spreading the material in the tank water. An 80% water change, re-dosed with 1.6 mL of TiO2. Fishes were fed twice (day 8 and 12) with a 1.5% body weight maintenance ration. This was done post water change but prior to re-dosing to minimise the risk of ingestion of TiO2 during feeding.
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
TiO2 bulk as dispersion
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
TiO2 bulk as dispersion
Basis for effect:
mortality
Details on results:
There was no overall material-type effect on animal behaviour.
Validity criteria fulfilled:
not specified
Conclusions:
No significant mortality of rainbow trout was observed at the single test concentration of bulk TiO2 (1 mg TiO2/L as dispersion) in a study similar to OECD 204.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12-16 March 1992
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: according to EPA-540/9-85-006, well documented study, no quantification of titanium concentration of test solutions
Qualifier:
according to
Guideline:
other: EPA-540/9-85-006, Acute Toxicity Test for Freshwater Fish
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): yes
Sonication/shaking/stirring of stock or test solution (duration): no
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: no information

Test solutions were prepared by weighing appropriate amounts of the test material into 15 liters of dilution water (originated from the Haskell Laboratory well) for immediate use.

DOC (well water) = 2.0 mg/L
Test organisms (species):
Pimephales promelas
Details on test organisms:
Fathead minnows ranged from 1.4 to 2.2 cm in standard length (mean 1.8 cm) and 0.0341 to 0.1876 g in weight (mean 0.0919 g).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
79 mg CaCO3/l
Test temperature:
21.3 °C (21.0-21.5°C)
pH:
7.4-7.8
Dissolved oxygen:
7.8-8.8 mg/l
Nominal and measured concentrations:
Nominal: 0 (control), 240, 340, 490, 700 and 1000 mg/l
Details on test conditions:
Glass aquaria (20 l) containing 15 l of test solution (approximately 19 cm liquid depth) were employed.
10 fish in each vessel.
Unaerated.
Not fed 48 hours prior to nor during the test.
16h light / 8h darkness.
Daily observations for mortality and effects.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
"Ti-Pure" Titanium dioxide as dispersion
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
"Ti-Pure" Titanium dioxide as dispersion
Basis for effect:
mortality
Validity criteria fulfilled:
not specified
Conclusions:
Less than 50% mortality of Pimephales promelas was observed at the highest test concentration (1000 mg TiO2/L as dispersion) in an acute GLP & guideline conform test.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
19-23 March 1992
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: according to EPA-540/9-85-006, well documented study, no quantification of titanium concentration of test solutions
Qualifier:
according to
Guideline:
other: EPA-540/9-85-006, Acute Toxicity Test for Freshwater Fish
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): yes
Sonication/shaking/stirring of stock or test solution (duration): no
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: no information

Test solutions were prepared by pipetting appropriate amounts of the test material into 15 liters of dilution water (originated from the Haskell Laboratory well) for immediate use.
DOC (well water) = 2.8 mg/l
Test organisms (species):
Pimephales promelas
Details on test organisms:
Fathead minnows ranged from 2.0 to 3.2 cm in standard length (mean 2.53 cm) and 0.1302 to 0.3898 g in weight (mean 0.2370 g).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
85 mg CaCO3/l
Test temperature:
20.1°C (19.3-20.8°C)
pH:
7.4-7.6
Dissolved oxygen:
7.4-8.8 mg/l
Nominal and measured concentrations:
Nominal: 0 (control), 240, 340, 490, 700 and 1000 mg/l
Details on test conditions:
Glass aquaria (20 l) containing 15 l of test solution (approximately 19 cm liquid depth) were employed.
10 fish in each vessel.
Unaerated.
Not fed 48 hours prior to nor during the test.
16h light / 8h darkness.
Daily observations for mortality and effects.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
"Ti-pure" Titanium dioxide RPS as dispersion
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
"Ti-pure" Titanium dioxide RPS as dispersion
Basis for effect:
mortality
Details on results:
All test solutions with TiO2 were cloudy, which prevented normal observation of the fish during the experiment.
Validity criteria fulfilled:
not specified
Conclusions:
Less than 50% mortality of Pimephales promelas was observed at the highest test concentration (1000 mg TiO2/L as dispersion) in an acute GLP & guideline conform test.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20-24 March 1992
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: according to EPA-540/9-85-006, well documented study, no quantification of titanium concentration of test solutions
Qualifier:
according to
Guideline:
other: EPA-540/9-85-006, Acute Toxicity Test for Freshwater Fish
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): yes
Sonication/shaking/stirring of stock or test solution (duration): no
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: no information

Test solutions were prepared by weighing appropriate amounts of the test material into 15 liters of dilution water (originated from the Haskell Laboratory well) for immediate use.
DOC (well water) = 2.8 mg/l
Test organisms (species):
Pimephales promelas
Details on test organisms:
Fathead minnows ranged from 1.3 to 2.7 cm in standard length (mean 198 cm) and 0.0293 to 0.1728 g in weight (mean 0.1021 g).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
79 mg CaCO3/l
Test temperature:
21.9 °C (21.7-22.0°C)
pH:
7.5-8.8
Dissolved oxygen:
7.3-7.7 mg/l
Nominal and measured concentrations:
Nominal: 0 (control), 240, 340, 490, 700 and 1000 mg/l
Details on test conditions:
Glass aquaria (20 l) containing 15 l of test solution (approximately 19 cm liquid depth) were employed.
10 fish in each vessel.
Unaerated.
Not fed 48 hours prior to nor during the test.
16h light / 8h darkness.
Daily observations for mortality and effects.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
"Ti-pure" Titanium dioxide R-931 as dispersion
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
"Ti-pure" Titanium dioxide R-931 as dispersion
Basis for effect:
mortality
Details on results:
All test solutions with TiO2 were milky, which prevented normal observation of the fish during the experiment.
Validity criteria fulfilled:
not specified
Conclusions:
Less than 50% mortality of Pimephales promelas was observed at the highest test concentration (1000 mg TiO2/L as dispersion) in an acute GLP & guideline conform test.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sub-chronic study; well performed and well documented with minor deviations
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)
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): via stock solution
Stirring of test solution (duration): yes in tanks during test
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance of solution: no information

10 g/L stock suspension of TiO2 was made by dispersing the test material in ultrapure (Millipore) water. Dosing was done after fish finished feeding to minimise ingestion of Ti02. After each water change, tanks were re-dosed with 80% of the nominated concentrations to bring the final tank concentration back up to the nominal dose (e.g. after an 80 % water change of the 1 mg/L tanks 20 % of the dose remained in the water so 1.6 ml of the 10 g/L stock was added to bring the total concentration back to 1 mg/L). After addition, water was stirred using a glass rod to ensure even mixing. Control tanks were also stirred with glass rods to standardise stress across all tanks.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Zebrafishs (n=360) were obtained from J&K Aquatics Ltd. in Taunton, Somerset, UK, Watchet, Somerset, UK
Fish were held in re-circulating, filtered, dechlorinated, Plymouth freshwater
Fish weighing 0.6±0.01 g (n=300)
Feeding: twice daily with brine shrimp
Acclimatisation period: 14d
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Test temperature:
25.8 ± 0.08 °C
pH:
7.45 ± 0.01
Dissolved oxygen:
7.92 ± 0.01 mg/l (>95% saturation)
Salinity:
ammonium, nitrite, nitrate
0.08±0.01, 0.17±0.02, <22 mg/L
Nominal and measured concentrations:
Nominal: 0 (control), 1.0 mg/L TiO2 bulk
Measured concentrations of titanium dioxide were 0.04 mg/L (control), 0.87 ± 0.01 mg/L bulk TiO2 in the treatments.
Details on test conditions:
TEST SYSTEM
- Test vessel: 20L glass tanks
- Renewal rate of test solution: 24h each with 80% water change
- Fish were fed with a single feed of brine shrimp prior to re-dosing the tanks with the appropriate TiO2 each day
- No. of organisms per vessel: 25
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

OTHER TEST CONDITIONS
- Photoperiod: 12:12 h
- Light intensity: laboratory light

TEST CONCENTRATIONS
- 2 respecitvely concentrations (0 (control), 1 mg/L for bulk TiO2)

An 80% water change, re-dosed with 1.6 mL of TiO2.
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.87 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
TiO2 bulk powder as dispersion
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 0.87 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
TiO2 bulk powder as dispersion
Basis for effect:
mortality
Validity criteria fulfilled:
not specified
Conclusions:
No significant mortality of Danio rerio was observed at the single test concentration of bulk TiO2 (1 mg TiO2/L as dispersion) in a prolonged toxicity test similar to OECD TG 204.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2007-03-19 to 2007-04-13
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, no quantification of titanium concentration of test solutions
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OSPARCOM (2005-11), Protocol for a fish acute toxicity test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): no
Stirring of test solution (duration): 20 h
Solvents: no
Filtration or other separation of test material from test solution: yes, siphoning of so-called water accomodated fractions (WAFs)
Appearance of solution: no information

- Titanium dioxide was added to the test system via WAFs. Solutions of 4 litres were prepared, stirred for 20 hours, left undisturbed for 1 h. Afterwards, the solution containg the readily soluble material was siphoned into the test vessels (with some inconsistencies regarding the stock & WAF loading).
Details on test organisms:
- Test organisms were reared at Opus Plus Ltd and were 91 days old before test commencement.
- Length: 1.63 – 2.48
- The test organisms were reared and maintained at Opus Plus Ltd. Sheepshead minnow were held under flow-through conditions in 50 liter capacity glass tanks, containing 1 µm filtered ultra-violet treated seawater and supplied with gentle aeration. Water quality measurements and mortality
observations were conducted daily and fed food in flake form ad libitum at least twice daily. Fish were randomly selected for testing.
Test type:
semi-static
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
20.1-21.6°C
pH:
7.76-8.11
Dissolved oxygen:
90-97%
Salinity:
36‰
Nominal and measured concentrations:
10000 mg/l
Details on test conditions:
EXPERIMENTAL PORCEDURE
- The tests were conducted in a temperature controlled room to maintain 20±2 ºC in the test medium.
- The tests were conducted in 15 litre capacity glass tanks containing 3 litres of test media. The preparations were mixed in situ to ensure thorough dissolution before introduction of the test organisms.
- Gentle aeration was supplied to all tanks.
- ten fish exposed per concentration.
- Where carrier solvents were used, an additional solvent control vessel was tested. Limit tests were conducted under semi-static conditions (with
replacement of media after 48h) over 96h with ten fish exposed to the single concentration along with a control.
- All vessels were checked at 24h intervals to monitor the condition of test animals. Animals which appeared immobile were gently prodded, and those which showed no response or opercular movement were recorded as dead. Measurement of water quality (dissolved oxygen, pH, temperature and salinity) are carried out at each concentration prior to and following each renewal of media.


TEST MATERIAL PREPARATION
- Test materials were assessed for risk to health, and appropriate handling and containment procedures implemented. Comparisons of the reported and observed physical characteristics (eg form, colour, odour and pH) of the test material was made.
- The test materials were characterised following Opus Plus SOP 402, where a 1000 mg.l-1 stock was prepared and stirred for approximately 20 hours. This preparation was then left undisturbed for approximately one hour before a behaviour in seawater classification was assigned and subsequently prepared for testing according to Opus Plus SOP 403. Readily soluble materials were added directly or via a volume of treated seawater to the appropriate tank in the required quantity. Poorly soluble materials were prepared as water accommodated fractions (WAFs). Solutions of 4 litres
were prepared, stirred for 20 hours and then siphoned into the test vessels.


SEAWATER SOURCE
- Natural local seawater is pumped to holding tanks from Scapa Flow, Orkney.
- The treatment process was: sand filtered to remove particulate, filtered to 1µm, heated to 16 ºC, UV treated, degassed and held at a quantity of 3000 to 5000 litres. The water is constantly pumped through the system and trickle replenished as the water is used.
Reference substance (positive control):
no
Duration:
96 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
Details on results:
The 96h limit test met all relevant validity criteria. Titanium dioxide exhibited no effect at 10000 mg/L after 96h (tested as so-called water accommodated fractions (WAFs) to the marine fish Cyprinodon variegatus.
Reported statistics and error estimates:
Mortalities were determined in each vessel every 24h period. The number of dead organisms were expressed as a proportion of the total number exposed. Where sufficient response was observed for range-finding and definitive tests, the 24h, 48h, 72h and 96h LC50 values, 96h LC90 and 96h NOEC values were calculated from the proportional response data using an appropriate method (ToxCalc V5, Tidepool Scientific Software).
Validity criteria fulfilled:
yes
Conclusions:
Less than 50% mortality of Cyprinodon variegatus was observed at the highest test concentration (10000 mg TiO2/L as so-called water accomodated fraction) in a GLP and guideline conform 96-h limit test.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 16-20 2006
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: According to OECD 203, well documented, no quantification of titanium concentration of test solutions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
10-fold increase in concentrations, 5 fish per concentration
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Direct addition of test material into test vessel (dispersion): yes
Stirring of test solution (duration): 1 h
Solvents: no
Filtration or other separation of test material from test solution: no
Appearance:
Controls and 0.1 mg/l test concentrations: clear and colorless with no precipitate at test start.
1 mg/l test concentrations: cloudy, with a slight amount of suspended substance present at the start.
10 and 100 mg/l test concentrations: cloudy (white in color) with suspended substance present at the start.

Test solutions were prepared by direct addition of the test material to Haskell Laboratory well water (HLWW) (15 l) and stirring for approximately 60 minutes. HLWW is aerated, passed through a green sand filter to remove iron, and filtered through 50-, 10-, and 0.45 µm filters to remove particulates. The water is heated or chilled as appropriate and distributed through aged PVC-piping.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Rainbow trout (Oncorhynchus mykiss) were purchased from Thomas Fish Company, Anderson, California.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
100-140 mg CaCO3/l
Test temperature:
12.2°C (range 12.1-12.3°C)
pH:
7.4-7.8
Dissolved oxygen:
9.5-10.6 mg/l
Nominal and measured concentrations:
Nominal: 0 (control), 0.1, 1, 10 and 100 mg/l.
Details on test conditions:
Test chambers were stainless steel aquaria (30*30*30 cm) which held approximately 15 l of test solution (test solution depth: 17.5 cm). 5 fish in each test chamber. Each chamber was covered with a glass plate to prevent the fish from escaping.
16 hours light / 8 hours darkness.
Test solutions were not aerated during the test.
Mortality and behavioral observations were made every 24 hours and at test end. Criteria for death were the absence of opercular movement and lack of reaction to gentle prodding.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
TiO2 R101 Pigment Grade as dispersion
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
TiO2 R101 Pigment Grade as dispersion
Basis for effect:
mortality
Validity criteria fulfilled:
yes
Conclusions:
Less than 50% mortality of rainbow trout was observed at the highest test concentration (100 mg TiO2/L as dispersion) in an acute toxicity test equivalent to OECD Guideline 203.

Description of key information

 Dispersions of microsized TiO2 are not acutely toxic to fish up to at least 1000 mg/L (LC50 > 1000 mg/L) and up to 10,000 mg/L (LC50: > 10,000 mg/L) in fresh- and saltwater, respectively. Considering the low solubility of microsized TiO2, it is concluded that microsized TiO2 is not acutely toxic up to its solubility limit. Dispersed nanosized TiO2 is not acutely toxic to fish up to at least 100 mg/L (LC50 > 100 mg/L) in freshwater.

Key value for chemical safety assessment

Additional information

Microsized TiO2:

Unbounded 96 h LC50 values of > 1000 mg/L (nominal) for Pimephales promelas and > 100 mg/L (nominal) for Oncorhynchus mykiss in freshwater were derived in acute toxicity tests of dispersed microsized titanium dioxide (Hutton et al. 1992a-c, Turner et al. 2006). Sub-chronic toxicity tests with dispersed microsized titanium dioxide generated unbounded 14 d LC50 values of > 0.87 and > 1.1 mg/L (measured) for Oncorhynchus mykiss and Danio rerio in freshwater (Boyle et al. 2013, Ramsden et al. 2013). One supporting study by Wyrwoll et al (2014) on fish embryos points to a lack of acute toxicity of dispersed microsized TiO2 up to 100 mg/L (72 h LC50 > 100 mg/L). An unbounded 96 h LC50 of > 10,000 mg/L (nominal) was observed for Cyprinodon variegatus exposed to dispersed microsized TiO2 in saltwater (Thomson et al. 2007).

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, the acute fish experiments 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 fish up to its solubility limit.

 

Nanosized TiO2:

Unbounded 96 h LC50 values of > 100 mg/L (nominal) were reported for the toxicity of nanosized TiO2 dispersions and Carassius auratus and Oncorhynchus mykiss (Ates et al. 2013, Turner et al. 2006). Furthermore, unbounded 96 h LC50s of > 1 mg TiO2/L (measured) were derived for Oncorhynchus mykiss and Danio rerio exposed to dispersed nanosized TiO2 for 14 days in three sub-chronic studies (Boyle et al. 2013, Federici et al. 2007, and Ramsden et al. 2013). Acute toxicity tests with embryos of Danio rerio generated 72 h and 96 h LC50 values of > 100 mg/L (nominal) for nanosized TiO2 dispersions (Wyrwoll et al. 2014, Clemente et al. 2014).

Four supporting studies further indicate that nano-TiO2 dispersions are not acutely toxic to freshwater fish up to at least 100 mg/L since LC50 values ranged from > 10 mg/L to at least > 100 mg/L (nominal, dispersed) for adult and embryonic life stages of Danio rerio and Oryzias latipes (Griffit et al. 2008, Li et al. 2014, Yang et al. 2013, and Ma et al. 2012). However, the latter tests were performed with inappropriate life stages and for a too short duration (Li et al. 2014), the provided information on methods and validity was not sufficient (Yang et al. 2013), or tests were performed at a water hardness that does not meet guideline criteria (Ma et al. 2012).

Marine data do not exist.

Based on a weight of evidence approach, dispersed nano- and microsized TiO2 is not acutely toxic to fish since unbounded LC50 values of > 100 mg and > 1000 mg TiO2/L were derived in freshwater, respectively. Further, an unbounded LC50 value of > 10000 mg/L for dispersed microsized TiO2 in saltwater points to a low potential for acute toxicity to fish.Considering the low solubility of microsized TiO2, it is further concluded that microsized TiO2 is not acutely toxic up to its solubility limit.