Reaction mass of disodium hexatitanium tridecaoxide and disodium trititanium heptaoxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-06-15 to 2010-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP Guideline study reliable without restrictions

Justification for type of information:

Disodium titanate substance (EC 234-802-9) has the molecular formula Na2TiO3 and its composition is expressed as (Na2O)x(TiO2), where x is ranging from 0.1 to 6 according to the SIP. This substance, Reaction mass of Disodium Hexatitanate and Sodium Metatitanate, has a value of x = 0.21, calculated from XRF results, has been identified as a mixture of two specific types of disodium titanate and is therefore within the scope of the disodium titanate SIP. It is assessed therefore that disodium titanate is an acceptable read-across substance for Reaction mass of Disodium Hexatitanate and Sodium Metatitanate

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed, 2009-03-04

Analytical monitoring:

yes

Details on sampling:

- Sampling for Chemical Analysis:
At the beginning of the test, duplicate samples of each of the test solutions and the control were taken. At the end of the test, immediately after determination of the biological and physico-chemical parameters, the test solutions of the replicates of each treatment were mixed. Duplicate samples were taken from the control, each of the test solutions and from the test vessels without algae with the highest test item concentration. Immediately after sampling and before shipment, the samples were stored in polyethylene bottles in the refrigerator.

- Sample Preparation / Analysis:
All samples received were stored refrigerated at test site up to sample preparation. Water samples were filtered (0.45 µm pore size), the filtrate acidified 1v/v (65% HNO3) and directly used for ICP - OES analysis to determine the content of dissolved titanium.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
- Preparation of the Test Solutions:
A stock solution (water-accommodated fraction, WAF) at a loading rate of nominal 100 mg test item/L was prepared by dissolving 100 mg of the test item in 1 L of test medium. This stock solution was agitated for seven days by using a horizontal laboratory shaker at ambient temperature in the dark. The shaker was set to a frequency of 100 rpm to keep the test item in permanent suspension according to OECD (2001). After that, the stock solution was filtered (glass fibre filter, pore size 0.4 µm). The filtrate was used to prepare the desired test item concentration.

- Controls: test organisms were exposed to controls without the test item (test concentrations: 0).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: SAG 61.81
- Source: Georg-August-Universität Göttingen, D-37073 Göttingen, Germany
- Age of pre-culture: 4 days

PRE-CULTURE CONDITIONS
To adjust the algae to the test conditions a pre-culture was inoculated by an agar stock culture and incubated under test conditions:
- pH-value: 6.0
- Number of cells per mL in the pre-culture before inoculating the test solution: 292.5 * 10E4
- Amount of liquid stock culture per pre-culture vessel: 100 mL
- Pre-culture vessels: 300 mL Erlenmeyer flasks
- Number of replicates: 2
- Light: light/dark (24/0 h); fluorescent tubes of universal white type
- Light intensity: 60 – 120 µE/m2 * s
- Shaker: 100 ± 5 oscillations/min
- Temperature in the test room: 21 - 24°C, controlled at ± 2°C

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

no

Test temperature:

22.0 to 23.6°C

pH:

pH values (pH0) at the beginning and pH values (pHe) at the end of the test

Treatment
[% of WAF] pH0 pHe
Control 6.0 6.2
100.0 6.1 6.2

Nominal and measured concentrations:

nominal conc.: 0 (control), water-accommodated fraction (WAF) at a loading rate of 100 mg/L
measured conc.: measured concentrations of titanium were below the limit of detection (LOD: 0.0027 mg/L regarding titanium determination) in all samples.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Volume of test solution per test vessel: 100 mL
- Renewal rate of test solution: none
- Initial cells density: 0.5 * 10E4 cells/mL
- Control end cells density (mean): 137.2 * 10E4 cell numbers/mL
- Number of replicates per test item concentration: 6
- Number of replicates in the control: 6
- Number of replicates for stability check (limit concentration without algae): 2

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: To optimise growth of Pseudokirchneriella subcapitata the test was performed with an algal medium recommended for unicellular green algae by “Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Unversität Göttingen (SAG) – List of Strains” (Schlösser 1982). The medium has been proved suitable for the maintenance of cultures at the SAG for many years. The algal medium (Kuhl and Lorenzen, 1964) consists of trace elements and macronutrients in the following final concentrations [mg/L]:

KNO3 1011.1
NaH2PO4 * 1 H2O 621.0
Na2HPO4 * 2 H2O 89.0
MgSO4 * 7 H2O 246.5
CaCl2 * 2 H2O 14.7
FeSO4 * 7 H2O 6.9
Titration complex III 9.3
(EDTA disodium salt dihydrate)
H3BO3 0.061
MnSO4 * 1 H2O 0.169
ZnSO4 * 7 H2O 0.287
CuSO4 * 5 H2O 0.00249
(NH4)6Mo7O24 * 4 H2O 0.01235

There is no indication of interactions between this medium and the test item.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Intervals of water quality measurement: After temperature adaptation of the test solutions, pH and temperature (automatically at least once per hour throughout the test in a separate test vessel placed on the shaker containing approx. 100 mL deionised water) were measured and recorded for each test concentration and the control in 1 replicate. At the end of the test, the pH was measured and recorded in one replicate of the concentration and the control.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light regime: permanent, light/dark (24/0 h)
- Types of light: fluorescent tubes of universal white type (Osram Lumilux 58W/865)
- Light intensity: mean value of six measurements: 86.2 µE/m2s
- Determination of light intensity measurement: Photometer LI-250A with sensor LI-190SA, LI-COR GmbH, Bad Homburg, Germany
- Shaker: 100 ± 5 oscillations/min; the test vessels were placed randomly on the shaker

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Growth inhibition was determined daily by determining the number of cells per volume test solution. The cell concentration of each test vessel was determined and recorded daily for 3 days after beginning of the test (THOMA Counting Chamber with Microscope Ortholux II, Ernst Leitz KG, Wetzlar, Germany; Multiple Reader Tecan ULTRA, Tecan Deutschland GmbH, Crailsheim, Germany). Morphological deviations compared to the control cells were recorded at the end of the study.
- Method for Counting Cell Numbers: After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 µL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA). The results [RFU, relative fluorescence units] were converted into biomass concentration [cells/mL] using a calibration line. A dilution series of the pre-culture was used to determine the calibration line by comparison of cell numbers (counted using a microscope) and fluorescence intensity (measured using a fluorometer) in consideration of background fluorescence of the blank algal medium.

TEST CONCENTRATIONS
- Range finding study: Based on the results of a preliminary non-GLP range finding test, where no effects were found at 0.1, 1, 5, 25, and 100% of the water-accommodated fraction at a loading rate of 100 mg test item/L, a limit test was performed using the water-accommodated fraction (WAF) at a loading rate of 100 mg test item/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % of WAF

Nominal / measured:

nominal

Conc. based on:

other: WAF at a loading rate of 100 mg/L

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: % of WAF

Nominal / measured:

nominal

Conc. based on:

other: WAF at a loading rate of 100 mg/L

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % of WAF

Nominal / measured:

nominal

Conc. based on:

other: WAF at a loading rate of 100 mg/L

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: % of WAF

Nominal / measured:

nominal

Conc. based on:

other: WAF at a loading rate of 100 mg/L

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities

Results with reference substance (positive control):

- Results with reference substance valid: yes
The reference item potassium dichromate (K2Cr2O7) is tested at least twice a year in order to determine the sensitivity of the organisms from the culture, and to demonstrate that the test conditions are appropriate. Results of the latest reference test performed in January 2010, the index 50 indicates 50% growth inhibition compared to the control:

Yield: EC50 (72 h): 0.23 mg/L, growth rate: EC50 (72 h): 0.85 mg/L.

Based on an international ring test (ISO 2004) mentioned in OECD guideline 201, the ErC50 (72h)-values for potassium dichromate obtained from different laboratories should be 1.19 mg/L with a standard deviation of 0.27 mg/L. The EC50 value for the toxic reference item, potassium dichromate, was determined as 0.85 mg/L. This value is within the historical range of the results of our laboratory (mean EC50 value = 1.15 ± 0.42 mg/L) and within the upper (2.54 mg/L) and lower (0.44 mg/L) warning limit. The warning limits were calculated as two standard deviations from the historical mean of the EC50 (all calculated using the log concentration; according to Environment Canada 2005). Furthermore, the EC50 value is within the refined range of 1.10 ± 0.48 mg/L, recommended based on an international ring test (Pattard et al. 2009). Therefore, the results of this reference test are acceptable and the test conditions are reliable.

Reported statistics and error estimates:

The data were evaluated for homogeneity of variances by Levene's Test and Student t-test to calculate whether there were significant differences between the growth of algae in the controls and the algae exposed to the test item concentration. The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

Validity criteria fulfilled:

yes

Conclusions:

No statistically significant differences were found between the control and the water-accommodated fraction (WAF) at a loading rate of 100 mg test item/L at p ≤ 0.05. The EC50 (72 h) was therefore determined to be higher than the tested concentration. The results of the study demonstrate that the no-observed effect concentration (NOEC) for Pseudokirchneriella subcapitata for the parameters yield and growth rate is above the water-accommodated fraction (WAF) at a loading rate of 100 mg test item/L (nominal concentration).

Description of key information

No statistically significant differences were found between the control and the water-accommodated fraction (WAF) at a loading rate of 100 mg test item/L at p ≤ 0.05. The EC50 (72 h) for Disodium titanate was therefore determined to be higher than the tested concentration.

The results of the study demonstrate that the no-observed effect concentration (NOEC) for Pseudokirchneriella subcapitata for the parameters yield and growth rate is above the water-accommodated fraction (WAF) at a loading rate of 100 mg test item/L (nominal concentration).

Additional information

No statistically significant differences were found between the control and the water-accommodated fraction (WAF) at a loading rate of 100 mg test item/L at p ≤ 0.05. The EC50 (72 h) for Disodium titanate was therefore determined to be higher than the tested concentration.

The results of the study demonstrate that the no-observed effect concentration (NOEC) for Pseudokirchneriella subcapitata for the parameters yield and growth rate is above the water-accommodated fraction (WAF) at a loading rate of 100 mg test item/L (nominal concentration).