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

ACCLIMATION
- Acclimation period: 5 days before treatment
- Acclimation conditions: the egg masses were taken from the cultures and placed in small aerated vessels with test water at about 20 °C.
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Post exposure observation period:
No post-exposure observation period was performed.
Hardness:
P25:
Test start: 110 – 150 mg/L as CaCO3 equivalents
Test end: 210 mg/L as CaCO3 equivalents in one representative replicate (100 – 120 mg/L as CaCO3 equivalents in the controls)
NM-101:
Test start: 140 mg/L CaCO3 equivalents in one representative replicate (130 – 150 mg/L CaCO3 equivalents in the control)
Test end: 170 mg/L CaCO3 equivalents in one representative replicate (150 – 170 mg/L CaCO3 equivalents in the control)
Test temperature:
20 ± 2°C
pH:
permitted range: pH 6 – 9
Dissolved oxygen:
About 100% at test start and test end (demanded threshold value: 60%)
Salinity:
not required
Ammonia:
P25:
Test start: 0.2 highest test concentration (0.8 - 1.0 (control)
Test end: 9.2 highest test concentration (8.0 - 10.2 control)
NM-101
Test start: 0.2 highest test concentration (0.5 - 0.9 control)
Test end: 0.6 highest test concentration (0.1 - 7.5 control)
Nominal and measured concentrations:
nominal concentrations: 15, 23, 39, 63, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test container (material, size): round glass beaker
- Sediment volume: 3 L
- Weight of sediment: 370 g dry mater
- Depth of sediment and overlying water: 2 cm sediment and 8 cm overlaying water
- Aeration: yes
- Aeration frequency and intensity: gentle

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

OVERLYING WATER CHARACTERISTCS
- Type of water: purified tap water

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

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

TITANIUM CONCENTRATIONS

P25 - Ti concentrations in overlaying water:

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

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

Hombikat UV 100 - Ti concentrations in overlaying water:

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

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

Hombikat UV 100 - sediment concentrations:

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

DLS RESULTS (Z-average)

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

- Hombikat UV 100: no measurements performed

Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the nano sized TiO2 materials P25 (anatase, rutile, 21 nm) and Homibkat UV 100 (anatase, 8 nm), which were applied via the overlaying water to the test system were tested in a chronic sediment toxicity test according to OECD 219. Both materials did not affect the emergence and development rate of Chironomus riparius, resulting in 28 d-NOEC values of ≥ 100 mg n-TiO2/L (nominal). For Homibkat UV 100 all validity criteria were fulfilled. The mean emergence in the controls of the P25 test was 68% at test end. The miniumum as stated in the guideline is 70%. As deviation is only minor and all other validity criteria are fullfilled and P25 induced any effects, this test was not repeated.
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Sediment and pore water were not analysed; overlaying water was spiked and analysed.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 225 (Sediment-Water Lumriculus Toxicity Test Using Spiked Sediment)
Deviations:
yes
Remarks:
exposure via the water phase and not via the sediment
Principles of method if other than guideline:
The test substance was applied to the overlaying water and was not spiked directly to the sediment as recommended in the guideline. Two experiments were performed, in the first experiment concentrations between 15-100 mg TiO2/L were tested and in the second experiment only 100 mg TiO2/L was tested. Only in the second experiment TiO2 concentration in the overlaying water were analysed.
GLP compliance:
no
Remarks:
But the test followed GLP principles (laboratory equipment was controlled and protocolled according to GLP, no quality assurance unit involved).
Analytical monitoring:
yes
Details on sampling:
SEDIMENT
Sediment was not included in analytical monitoring.

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

PREPARATION OF STOCK DISPERSIONS:
- Direct addition of test material into test vessel: no
- Sonication of stock dispersion: The desired test amount was weighed into a glass vessel containing the appropriate volume of dionized water. Dispersions were stirred for 60 seconds on a magnetic stirrer (900 rpm) and placed into an ultrasonic water bath filled to one third of the dispersion height in the bottles (Badelin Sonorex RK 514 BH; 35 kHz; 215/860 W) for three minutes.
- Solvent: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of test dispersion: no information
- Renewal of test dispersion: no, static
- Test medium: Reconstituted freshwater (OECD 203)
Test organisms (species):
Lumbriculus variegatus
Details on test organisms:
TEST ORGANISM
- Common name: blackworm
- Source: Fischfutter Etzbach (Mechernich-Bergheim, Germany)
- Food type: the worms were fed with fish food suspension (50 g/L TetraMin ® )
- Frequency:daily
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
yes
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
178 – 267 mg/L CaCO 3
Test temperature:
20 ± 2°C
pH:
7.5 – 8.0
Ammonia:
0.2 - 1.4
Nominal and measured concentrations:
nominal concentrations (sediment overlying water): 15; 23; 39; 63 and 100 mg NM-105/L
Details on test conditions:
TEST SYSTEM
- Test container (material, size): glas vessels, 250 mL total volume with plastic lid
- Weight of wet sediment: 80 g wet weight
- Overlying water volume: 180 mL
- Depth of sediment and overlying water: ca. 1.5 cm height of sediment in test vessel
- Aeration: yes
- Aeration frequency and intensity: continuous aeration
- Replacement of evaporated test water, if any: static; 3 days per week adjustment for evaporated test medium

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

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

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

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

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

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

Validity criteria fulfilled:
yes
Conclusions:
Dispersions of the nano sized TiO2 material P25 (21 nm) were directly added to the overlaying water phase in a chronic sediment toxicity test according to OECD guideline 225. The derived 28-d NOEC values for biomass and reproduction of Lumbriculus variegatus were ≥ 100 mg/L (nominal). Chemical analysis confirmed that 110% of the nominal test concentration was applied to the overlaying water and that around 99% of the nominal concentration was removed from the water column after 1 d of exposure.
Endpoint:
sediment toxicity: long-term
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
March 2008
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study conducted according to ASTM E1706 guideline, however exposure concentrations were not measured.
Qualifier:
according to
Guideline:
other: ASTM E1706
Deviations:
not specified
GLP compliance:
no
Remarks:
Compliance statement to ASTM E1706 guideline attached (see below)
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Hyalella azteca
Details on test organisms:
TEST ORGANISM
- Age of animals at beginning of exposure: 5-7 days
- Feeding during test
- Frequency: each day

ACCLIMATION
- Acclimation period: no
Study type:
laboratory study
Test type:
semi-static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
125 mg/L CaC03
Test temperature:
Test was performed in a thermostatically controlled room at 25°C
pH:
measured at the beginning of the test, after 21 days and at the end of the test:
Exxaro SO4:
day 0: range 7.30-7.32
day 21: range 7.33-7.40
day 28: range 7.22 -7.55

RBM SO4:
day 0: range 7.28-7.38
day 21: range 7.25-7.41
day 28: range 7.38-7.44
Dissolved oxygen:
measured at the beginning of the test, after 21 days and at the end of the test:
Exxaro SO4:
day 0: range 7.65-7.88 mg/L
day 21: range 4.89-5.64 mg/L
day 28: range 4.28-5.43 mg/L

RBM SO4:
day 0: range 7.68-7.83 mg/L
day 21: range 4.89-5.66 mg/L
day 28: range 4.54-4.90 mg/L
Salinity:
Not measured
Ammonia:
No data
Nominal and measured concentrations:
Nominal test concentrations: control, 0.1; 0.3; 1; 3; and 10%
Details on test conditions:
TEST SYSTEM
- Test container (material, size): WECK container 500 mL
- Weight of sediment: 100 g per container

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 5
- No. of replicates per control / vehicle control: 5
- Feeding regime: each day

RENEWAL OF OVERLYING WATER
2 times a week

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): Borgman medium

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- Particle size distribution
- % sand: 75
- % peat: 5
- % clay: 20
- pH pore water: between 6.5 and 7.5

OTHER TEST CONDITIONS
- Photoperiod: 16h light/8h dark
Reference substance (positive control):
not specified
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 other: %
Nominal / measured:
nominal
Conc. based on:
other: test mat. Exxaro SO4
Basis for effect:
other: survival
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 other: %
Nominal / measured:
nominal
Conc. based on:
other: test mat. Exxaro SO4
Basis for effect:
other: growth expressed in µg DW/Hyalella
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 other: %
Nominal / measured:
nominal
Conc. based on:
other: test mat. RBM SO4
Basis for effect:
other: survival
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 other: %
Nominal / measured:
nominal
Conc. based on:
other: test mat. RBM SO4
Basis for effect:
other: growth expressed in µg DW/Hyalella
Reported statistics and error estimates:
Percentage survival was calculated as the mean of the 5 replicates. The growth was statistically determined by the dunnet test, comparison with control.

Exxaro SO4:

Overview % survival and growth (in dry weight µg/Hyalella) of Hyalella azteca after 28 days:

  % Survival  Growth (µg DW/Hyalella) 
 Control  92 272 
 Exxaro SO4 10%  98 284
 Exxaro SO4 3%  96 207
 Exxaro SO4 1%  96 271 
 Exxaro SO4 0.3%  98 263 
 Exxaro SO4 0.1%  96 256

No significant differences were observed at all test concentrations for Exxaro SO4.

RBM SO4:

Overview % survival and growth (in dry weight µg/Hyalella) of Hyalella azteca after 28 days:

  % Survival  Growth (µg DW/Hyalella) 
 Control 92 272 
 RBM SO4 10% 92 278
 RBM SO4 3% 98 220
 RBM SO4 1% 100 218 
 RBM SO4 0.3% 100 275 
 RBM SO4 0.1% 94 290

No significant differences were observed at all test concentrations for RBM SO4.

Validity criteria fulfilled:
yes
Conclusions:
No chronic toxicity on survival or growth of H. azteca was observed at all test concentrations of Exxaro SO4 and RBM SO4. The 28-d NOEC was ≥ 10 %.
Upgraded ilmenites (UGI) consist primarily (> 80%) of a titanate phase i.e., Ti (as TiO2) with Fe, Al, Si, Mg and other metals. Transformation/dissolution tests were performed with UGI products by analysing total dissolved concentrations of the trace and minor elements Al, Co, Cr, Cu, Fe, Mn, Mo, Ni, Nb, Ti, V and Zn over periods up to 28 days at loadings of 100, 10 and 1 mg/L. The T/D data revealed that only Fe, Mn and V among the 12 metals dissolved to any significant extent (maximum increase in Fe, Mn and V concentrations 26.2, 8.9 and 3.5 µg/l, respectively). Within the limits of experimental error, the other metals remained refractory to release, none dissolving from any of the slags at any of the loadings. Because of the structural similarity and the low solubility of both UGI slags and TiO2, read-across for ecotoxicological hazard data from UGI to TiO2 is considered relevant.
Endpoint:
sediment toxicity: short-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February-March 2007
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, meets general quality standards. No analytical dose confirmation. However, the applied test material amount is documented.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OSPARCOM guidelines (1995) A sediment Bioassay using an amphipod corophium sp
Principles of method if other than guideline:
The method investigated the effect on survival of Corophium exposed for 10 d to sediments amended with the TiO2 material.
GLP compliance:
yes
Remarks:
However, no GLP certificate and GLP statements not signed by the study director and the quality assurance
Analytical monitoring:
not specified
Vehicle:
yes
Remarks:
dry sediment
Details on sediment and application:
- Titanium Dioxide was characterised as poorly soluble and therefore was added to the test system via dried sediment.
Test organisms (species):
Corophium volutator
Details on test organisms:
- Test organisms and sediment were collected on 23 January 2007 from a site in the Bay of Suckquoy, Toab in Orkney and received on site on 23 January 2007 and acclimated to laboratory conditions for 13 days prior to testing.

- Specimens were gently sieved from their native sediment and held in plastic containers (approximately 5l capacity) containing some ambient water, with a small amount of detritus, until transfer to the laboratory. On return to the laboratory, the Corophium were transferred in ambient water to polythene tanks of approximately 200 litre capacity, gentle aeration was supplied. The tanks were held in a controlled-temperature room at approximately 15±2 °C.

- The stock animals were gradually acclimated from the ambient salinity of less than 5‰ to that of undiluted seawater (approximately 35‰) in increments of approximately 10‰ per day. Once acclimation is complete, the stock tanks were maintained under semi-static conditions until the initiation of the test. The holding period in the laboratory is between 3 and 14 days. Sufficient detrital material is retained in each tank to provide food and some bottom cover, but not of a density that prevents daily observation of mortality and morbidity. Dead or impaired animals are removed when observed.

- Specimens of approximately 5 mm in body length (excluding rostrum) were used in the toxicity tests.
Study type:
laboratory study
Test type:
static
Water media type:
saltwater
Type of sediment:
natural sediment
Limit test:
no
Duration:
10 d
Exposure phase:
total exposure duration
Test temperature:
14.4-15.8°C
pH:
8.01-8.23
Dissolved oxygen:
91-99 %
Salinity:
33-39%
Nominal and measured concentrations:
Nominal target (wet weight) concentrations of 100, 320, 1000, 3200 and 10000 mg TiO2/kg wet weight
(Concentrations not measured, however applied amount documented: 0.00752 g; 0.07509 g; 0.24051 g; ,0.751g; 7.517 g TiO2 were applied to 750.30 g; 751.89 g; 751.73 g; 751.20 g; 751.75 g wet weight sediment.)
Details on test conditions:
TEST SEDIMENT
- The wet to dry ratio of sediment used in this study was 1.58 and this was used to correct nominal wet sediment concentrations to nominal dry sediment concentrations.
- Sediment for use in toxicity testing was collected from a point adjacent to the site of Corophium collection. On return to the laboratory, the sediment was wet-sieved through 0.6 mm mesh to remove larger infaunal organisms and Corophium within the test size range, and allowed to settle in tall polyethylene containers. After settling, the supernatant water was decanted and the sediment stored in a room at approximately 4±2 °C, until required for testing.
- Particle size analysis characterised the sediment as well-sorted, fine sand with a silt/clay content of 3.56% by weight. Median particle diameter was 151 µm. The organic material content was estimated from weight loss on ignition to be 1.34%.
- Immediately before the initiation of the test, the sediment was thoroughly homogenised and a representative sample was taken for wet and dry weight determination. The ratio of wet weight to dry weight was subsequently used to convert nominal exposure concentrations on a wet weight basis to nominal exposure concentrations on a dry weight basis.



TEST PROCEDURE
- Tests were conducted in 1 litre capacity glass beakers each containing 2 cm depth (approximately 150 ml) of amended sediment and 850 ml of overlying seawater (1 µm filtered ultra violet treated seawater). Test beakers were maintained under constant dim illumination in a controlled temperature room for a test period of 10 days.
- Three replicates were prepared for each test concentration; controls are replicated five times. The beakers were assigned positions within the test area, arranged in rows of three to five and spaced to maintain effective separation of different treatments. Each row was covered with a rectangular sheet of perspex perforated with a small hole above the centre of each beaker.
- Aeration was provided and a stream of air bubbles were released at a depth of approximately 6 cm.
- Twenty animals were added to receiving vessels in two groups of ten and then dispensed into a test vessel. Sixty organisms were exposed in total per concentration and one hundred in five vessels for controls. The final level of water in each test vessel was marked with a permanent marker.
- Any visible signs of animals on the sediment surface whether alive or dead, were recorded, if applicable. At the end of the test period, the contents of each beaker were gently sieved through a 0.6 mm mesh, and all surviving animals separated and counted. Missing animals were counted as dead; early mortalities will either have been consumed by survivors or have decomposed during the test period.


TEST MATERIAL PREPARATION
- The test material was assessed for risk to health, and appropriate handling and containing procedures were implemented. Following this, a comparison of the reported and observed physical characteristics (eg form, colour, odour, pH and density) of the test material was carried out.
- In order to determine an appropriate test preparation method, an assessment was made of its behaviour in seawater. 1000 mg.l-1 of stock was prepared in filtered seawater, and the resulting mixture was stirred for approximately 20-24 hours. The mixture was then left to settle for approximately 1h, and its behaviour assessed (SOP 402).
- Test material preparation was conducted in accordance with SOP 403. Materials which were assessed to be poorly soluble were prepared by mixing initially with a small quantity of dry sediment, using a solvent of low toxicity such as acetone, ethanol or triethylene glycol if necessary. The dried sediment was then incorporated with the wet sediment (in the mixing container). If a material was classified as soluble or dispersible it was dissolved in a small quantity of sea water and added directly to the mixing container, containing the wet sediment. The mixing vessels were then placed on a platform shaker at 150 rpm for 3 hours. After this period, the contents of each container were equally distributed between the replicate vessels, three for test concentrations and five for control vessels.
- Seawater for testing was supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 1 µm before being employed for testing.
Reference substance (positive control):
no
Duration:
10 d
Dose descriptor:
LC50
Effect conc.:
> 14 989 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
>= 14 989 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
The test was conducted in accordance with the study plan and met all relevant validity criteria. There were no interferences in this test.
Reported statistics and error estimates:
The number of animals responding after 10 days in each replicate are expressed as a proportion of the nominal number exposed (20). The 10 day LC50, LC90 and NOEC values are calculated using an appropriate statistical method from the ToxCalc Version 5 software.
Validity criteria fulfilled:
yes
Conclusions:
The marine amphipod Corophium volutator was exposed to a sediment which was spiked with a titanium dioxide material in a non-standardized toxicity assay, under GLP conditions for ten days. TiO2 did not affect the survival of the test organism resulting in a 10 d-NOEC value of≥ 14989.26 mg/kg dw (nominal). Exposure concentrations were not measured. However, the applied test substance amount and the weight of the sediment in each treatment group was documented.


Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)
Deviations:
no
Principles of method if other than guideline:
Range-finding tests were performed in artificial sediment as well as natural sediment.
GLP compliance:
yes (incl. certificate)
Remarks:
signed 31-05-2016 / Range-finder was non-GLP
Analytical monitoring:
yes
Details on sampling:
At test start (after an equilibration period of 48 - 96 hours after addition of the test item), samples of sediment and overlying water are removed from additional analytic vessels per treatment for the analytical determination of the concentration of the test substance in sediment and overlaying water in accordance with OECD TG 225. Samples of the overlying water and the sediment of at least one replicate are analyzed at the end of the test at each test concentration. Additionally, the concentration of the test substance in the pore water will be measured at the beginning of the test at each treatment level.
Vehicle:
no
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Details of spiking: The solid test item is applied as a mixture with quartz sand to the sediment. The sediment is spiked in bulk at each concentration level. Sediment is mixed thoroughly with the applied test item / quartz sand mixture by hand mixing. The respective subsamples of the spiked sediment are filled into the test vessels. The overlaying water is filled into the vessels carefully to avoid a sediment raise.
- Equilibration time: 2-4 days
- Controls: Sediment without addition of the test item
Test organisms (species):
Lumbriculus variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Blackworm
- Source: ECT Oekotoxikologie GmbH, 65439 Flörsheim a. M., Germany; Specimens used in the test are bred in the laboratory of the Fraunhofer IME
- Breeding conditions: According to Annex 5 of OECD 225: On a layer of quartz sand purified tap water is used. The worms were fed twice a week with a suspension of powdered TetraMin® Hauptfutter (Tetra Werke, Melle, Germany).
- Age of animals at beginning of exposure: Synchronised worms, after a regeneration period of 10 to 14 days
- Feeding during test: Since food is added to the sediment prior to application of the test substance, the worms are not fed additionally during the test.

ACCLIMATION
- Acclimation period: 10-14 days
- Acclimation conditions: same as test
- Type and amount of food: Suspension of powdered TetraMin® Hauptfutter (Tetra Werke, Melle, Germany).
- Feeding frequency: Twice a week
- Health during acclimation: After regenerating, intact complete worms, which are actively swimming or crawling upon a gentle mechanical stimulus, are used for the test
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Remarks:
The range-finding test was also performed with natural sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
Overlying water: 90-300 mg/L asCaCO3 (measurements will be submitted)
Test temperature:
20°C ± 2°C (measurements will be submitted)
pH:
7.0 ± 0.5 (measurements will be submitted)
Dissolved oxygen:
will be submitted
Ammonia:
will be submitted
Nominal and measured concentrations:
Nominal: 0 (control), 62.5, 125, 250, 500, 1000 mg TiO2/kg
Range-finding test: 0 (control), 10, 100, 1000 mg TiO2/kg
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Glass wide mouth bottles (200 mL, 5.5 cm Ø)
- Sediment volume: will be submitted
- Overlying water volume: will be submitted
- Depth of sediment and overlying water: Sediment layer: 1.5 – 3 cm; Overlying water: 6-12 cm
- Aeration: yes
- Aeration frequency and intensity: Aeration of the overlaying water will be provided through an e.g. glass pipette driven through the cap and fixed 2-3 cm
above the sediment layer (2 - 4 bubbles/second)

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 6
- Feeding regime: Since food is added to the sediment prior to application of the test substance, the worms are not fed additionally during the test.

OVERLYING WATER CHARACTERISTCS
- Type of water: Reconstituted water according to OECD Guideline 203

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 4-5 %
- Composition (if artificial substrate): Sphagnum peat, air-dried, finely ground (≤ 1 mm) (4-5 %), Kaolinite clay (Kaolinite content ≥ 30%), air-dried (20 %), Industrial quartz sand, air-dried (75-76 %), Powdered leaves of Stinging Nettle (Urtica sp.) or other suitable green plant meal, at 0.4 -0.5 % of sediment DM
- Method of preparation (if artificial substrate): Mixing
- Maturation of artificial substrate: no
- Moisture: Test substrate is wetted with deionised water to reach a water content of 30 % to 50 %
- pH whole sediment: 7.0 ± 0.5
- Total organic carbon (%): 2 ± 0.5

OTHER TEST CONDITIONS
- Light quality: will be submitted
- Photoperiod: 16 h light:8 h dark
- Light intensity: 100 – 500 lux

EFFECT PARAMETERS MEASURED:
- During the exposure, the test vessels are observed in order to assess visually any behavioural differences in the worms
- Total number of living and dead individuals; test end
- Biomass (dry weight); test end

TEST CONCENTRATIONS
- Spacing factor for test concentrations: will be submitted
- Range finding study: yes
- Test concentrations: 0 (control), 10, 100, 1000 mg TiO2/kg
- Results used to determine the conditions for the definitive study: will be submitted
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol (PCP)
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
total number of worms
Remarks on result:
other: Range-finding test in artificial sediment
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
total number of worms
Remarks on result:
other: Range-finding test in artificial sediment
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
worm dry weight
Remarks on result:
other: Range-finding test in artificial sediment
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
worm dry weight
Remarks on result:
other: Range-finding test in artificial sediment
Results with reference substance (positive control):
Measured acute, water-only toxicity of PCP is in the range of toxicity as reported in OECD guideline 225 and references therein.
Reported statistics and error estimates:
For the determination of NOEC values, Shapiro-Wilk´s Test on Normal Distribution, Levene´s Test on Variance Homogeneity (with Residuals), trend analysis by Contrasts (Monotonicity of Concentration/Response) and Dunnett`s Multiple t-test Procedure were applied. The computer program ToxRat was used for the evaluation of the test.

Additionaly, in a range-finding test conducted in natural sediment, the following effect concentrations were determined:

A 28 d NOEC ≥ 1000 mg TiO2/kg sediment dw (nominal) for both total number of worms and biomass (worm dry weight), as well as a 28 d LOEC > 1000 mg TiO2/kg sediment dw (nominal) for both total number of worms and biomass (worm dry weight).

Validity criteria fulfilled:
not specified
Remarks:
will be sumbitted; range finding tests: Average number of living worms per replicate increased by a factor of 3.0 (artificial sediment) or 2.5 (natural sediment)
Conclusions:
In two range-finding tests following OECD test guideline 225, Lumbriculus variegatus were exposed to TiO2 nanoparticles (TiO2-uf-2; primary particle size: 19 nm) directly spiked into the sediment for 28 d. Tests were conducted in artificial sediment and natural sediment. For both sediment types, an unbounded 28 d NOEC ≥ 1000 mg TiO2/kg sediment dw (nominal) and a 28 d LOEC > 1000 mg TiO2/kg sediment dw (nominal) were determined for both total number of worms and biomass (worm dry weight).
Results from the definitive study will be submitted, when the final report is available.

Description of key information

Sediment toxicity tests reveal that microsized TiO2 is not chronically toxic to sediment organisms up to concentrations of at least 100,000 and 14,989 mg/kg dw (nominal) in freshwater and marine sediment, respectively. A sediment-spiked toxicity test (range finder) and water-spiked sediment toxicity tests further point to a low toxic potential of nanosized TiO2 for freshwater epi- and endobenthic organisms, with NOEC values of ≥ 1000 mg/kg dw and ≥ 100 mg/L (nominal) for sediment- and water-spiked tests, respectively.

Key value for chemical safety assessment

Additional information

Microsized TiO2:

Data are available for freshwater and marine sediments. A 28 d exposure of upgraded ilmenite (>80% TiO2) up to 100,000 mg/kg dw did not affect survival and growth of Hyalella azteca in freshwater sediment (Arcadis et al. 2008).

The 10 day exposure of the marine amphipod Corophium volutator to a sediment spiked with microsized TiO2 in a non-standardized toxicity assay under GLP conditions did not affect its survival resulting in a 10-d NOEC of 14,989 mg/kg dw (nominal) (Hudson, 2007).

In a freshwater mesocosm study (Jovanovic et al. 2016), rated as supporting, autoclaved natural sediment was spiked with 25 mg/kg titanium dioxide E171 and incubated for 100 days (July to November) in the original pond. Thereafter, the reinvasion of macroinvertebrates was analysed by evaluating the macroinvertebrate community structure by means of non-metric multidimensional scaling (NMDS), multivariate analysis of variance, and indicator value analysis after identifying all recovered organisms at the lowest possible taxonomic level. Czekanowski’s index was 0.61, meaning that 39% of the macroinvertebrate community structure was negatively or positively affected by TiO2. The absolute abundance of Chironomus luridus agg, Radix auricularia, Theodoxus transversalis, Lymnea stagnalis, Psectrotanypus varius, Trichoptera, Endochironomus tendens, Chaoborus sp., Baetis sp., Chironomus plumosus, and Asellus aquaticus was comparable to the abundance in controls. In contrary, the absolute abundance of the gastropods P. corneus and R. labiata was significantly lower in the TiO2 treatment compared to control, and the absolute abundance of the Ceratogonidae (Diptera) per tray was significantly enhanced in the TiO2 treatment compared with the control. Nevertheless, this study is only considered supporting, since reinvasion or avoidance is generally not considered as a relevant endpoint for the environmental hazard assessment. Furthermore, it is not possible to derive valid effect values, since only one concentration was tested, and sediment and water were not well characterized.

Nanosized TiO2:

Regarding the toxicity of TiO2-NP to sediment organisms, a range-finding test conducted in the course of a GLP guideline study following OECD 225 revealed that the total number and biomass (worm dry weight) of Lumbriculus variegatus was not affected up to 1000 mg TiO2/kg sediment dw (28 d NOEC ≥ 1000 mg/kg dw, nominal) when directly spiked into artificial and natural sediment (Simon, 2017). Furthermore, reproduction and growth of Lumbriculus variegatus as well as emergence and development rate of Chironomus riparius were not affected up to 100 mg/L TiO2 (rutile/anatase, both tests: 28 d NOEC ≥ 100 mg/L) in water-borne exposure tests according to OECD 225 and OECD 219, respectively (Schäfers & Weil, 2013; Hund-Rinke & Klawonn, 2103). 

One supporting sediment toxicity study is available and results indicate that nanosized TiO2 is also not chronically toxic to Hyalella azteca when applied via the sediment: In a chronic toxicity test according to EPA 600/R-99/064, Wallis et al. (2014) exposed Hyalella azteca (7-8 d old) to 20 and 100 mg/L of the nano-sized TiO2 material P25 (nominal) in a natural sediment under laboratory light and simulated solar radiation conditions resulting in 21-d NOEC values for survival and growth rate of ≥ 100 mg/L sediment (nominal). However, the applied natural lake water was not well characterized by Wallis et al. (2014) and thus results are considered as supporting data.

In sum, micro- and nanosized TiO2 appear to have a low potential for acute and chronic freshwater and marine sediment toxicity based on data available for insect, crustacean and annelid species.