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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 06 July 2017. Experimental completion date: 31 July 2017
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Test substance not detected in test system
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: TXAX
Chemical Name: Potassium titanium oxide (K2Ti6O13)
CAS Number: 12056-51-8
Batch: MA-5X044
Purity: typical concentration 97.2% (w/w)
Physical state/Appearance: pale yellow powder
Expiry Date: 01 February 2020
Storage Conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and each test group for quantitative analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 5, 12 and 19 and of the expired test preparations from the pooled replicates on Days 3, 7, 14 and 21. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

The test samples were thawed with the aid of sonication. Nitric acid (2 mL) was added to a volume (100 mL) of sample and the samples were ultrasonicated for 15 minutes before being filtered through 0.45 µm cellulose acetate filters as shown in Table 1.
Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of deionised reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)
Discussion
Given that the test item was expected to be insoluble, it was evident from these results that centrifugation was not able to fully remove the undissolved test item.
Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L stirred via propeller stirrer at approximately 1500 rpm for 24 hours prior to removal of any undissolved test material by filtration using a 0.2 μm Sartorius Sartopore filter, first 2 liters discarded in order to pre-condition the filter.

Experimental Preparation
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartprius Sartopore filter (first approximate 2 liters discarded in order to pre condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 6.25, 12.5, 25 and 50% v/v saturated solution.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 3, 5, 7, 10, 12, 14, 19 and 21
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Modified ISO medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
The water hardness was observed to be in the range 230 to 258 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.
Test temperature:
Temperature was maintained at 21 °C to 22 °C throughout the test
pH:
7.5 - 8.0
There were no treatment related differences for pH
Dissolved oxygen:
7.8 - 9.1 mg O2/L
There were no treatment related differences for oxygen concentration
Salinity:
not applicable freshwater
Nominal and measured concentrations:
6.25, 12.5, 25, 50 and 100% v/v saturated solution (nominal).
Measured concentrations of the test item in the test solutions could not be determined
Details on test conditions:
Definitive Test
Due to the very low solubility of the test item in water, as determined in the preliminary media preparation trial, significant toxic effects were not expected and therefore a range finding test was considered unnecessary.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.

Exposure Conditions
For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed three times per week on Days 0, 3, 5, 7, 10, 12, 14, 17 and 19. The adult daphnia were transferred to fresh media by wide bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded
Each daphnid received approximately 5 or 15 µL of an algal suspension (Desmodesmus subspicatus) and 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
50 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
36 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
94 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
25 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
50 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Verification of Test Concentrations
Chemical analysis of the test preparations (see Annex 3) throughout the test duration showed measured test concentrations of less than the LOQ of the analytical method employed were obtained which was determined to be 0.0010 mg/L titanium, equivalent to 0.0021 mg/L test item. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
As measured concentrations of the test item in the test solutions could not be determined all results are based on nominal test concentrations.

Lethal Effects on the Parental Generation (P1)
Mortality (immobilization) occurred predominantly at the highest test concentration of 100% v/v saturated solution, resulting in 40% mortality by Day 21.
Mortality was also observed at the test concentrations of 6.25, 12.5 and 25% v/v saturated solution. However, statistical analysis of the mortality data using the Step-down Cochran-Armitage Test procedure showed that the observed mortalities were not significantly different (P≥0.05) when compared to the control group and are therefore treated as inadvertent mortalities.
No mortalities occurred at the 50% v/v saturated solution test group throughout the test.

Sub-lethal Effects on the Parental Generation (P1)
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P≥ 0.05) between the control and the 6.25 and 12.5% v/v saturated solution test groups in terms of length of the daphnids after 21 days exposure to the test item, however at test concentrations of 25, 50 and 100% v/v saturated solution significant differences (P<0.05) were observed.

Effects on Reproduction
After 21 days there were no statistically significant differences between the control and the 6.25, 12.5 and 25% v/v saturated solution test groups in terms of the number of live young produced per adult, however, the 50 and 100% v/v saturated solution test groups showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult.

Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. An assessment made at each media renewal showed the "filial" daphnids produced by the 6.25 and 25% v/v saturated solution test groups were in the same general condition as the young produced by the controls over the duration of the test, however, some young produced by the 12.5, 50 and 100% v/v saturated solution test groups, were observed to be pale when compared to the control young on Days 9, 10, 11, 13 and 19.
Young were first produced in the control test group on Day 8 of the test.
There were no unhatched eggs or dead young recorded in all control and treatment groups throughout the test.

Lowest Observed Effect Concentration
The LOEC was 50% v/v saturated solution as this test group produced significantly fewer live young per adult (P<0.05) than the control group.

No Observed Effect Concentration
The NOEC was 25% v/v saturated solution as there were no significant mortalities (immobilization) observed in the parental generation (P1) and there were no significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

The following ECx(immobilization) values based on nominal test concentrations was estimated by inspection of the data at 21 days:

Endpoint Concentration
(% v/v Saturated Solution)
Immobilization EC50 >100
95% confidence limits Not determined 
No Observed Effect Concentration  50
Lowest Observed Effect Concentration  100

The following ECx(reproduction) values based on nominal test concentrations were calculated by the Maximum‑Likelihood Probit method (Finney 1971) at 21 days:

Endpoint Concentration
(% v/v Saturated Solution)
Reproduction EC10 36
95% confidence limits 20 to 65
EC50 94
95% confidence limits 42 to >100
No Observed Effect Concentration 25
Lowest Observed Effect Concentration  50

 Validation Criteria

The following validation criteria were achieved during the test:

Required Actual
Control mortality 20% 0%
Mean number of live young per surviving adult (control group) 60 after 21 days 110
Coefficient of variation for Control group * £25% 17.60%
Ephippia produced in the control group 0 0
Dissolved oxygen >3 mg O2/L ≥7.8 mg O2/L
pH (control group) 6 to 9 7.4 to 8.1
Variation  1.5 0.7

*=   Based on total number of living offspring per parent animal alive at the end of the test

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item resulted in significant mortalities at the concentration of 100% v/v saturated solution resulting in 40% mortalities by Day 21.
The 21 Day EC50 (immobilization) value, based on nominal concentrations, for the parental daphnia generation (P1) was estimated to be greater than 100% v/v saturated solution.
The 21 Day EC50 (reproduction) based on nominal concentrations was 94% v/v saturated solution with 95% confidence limits of 42 to greater than 100% v/v saturated solution.
The LOEC and the NOEC based on nominal were 50 and 25% v/v saturated solution respectively.
Executive summary:

Introduction

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "DaphniamagnaReproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0010 mg/L titanium, equivalent to 0.0021 mg/L test item was obtained from a saturated solution method of preparation.

Daphnia magnawere exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution for a period of 21 days. The test solutions were renewed 3 times per week throughout the test. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate two liters discarded in order to pre-condition the filter) This saturated solution was then further diluted as necessary, to provide the remaining test groups.

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin®flake food suspension.

Results

Chemical analysis of the test preparations throughout the test period showed measured test concentrations of less than the LOQ of the analytical method employed were obtained which was determined to be 0.0010 mg/Ltitanium, equivalent to 0.0021 mg/L test item. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Exposure of Daphnia magnato the test item gave the following results based on the nominal test concentrations:

Endpoint

Concentration
(% v/v Saturated Solution)

Immobilization

EC50

>100

95% confidence limits

Not determined

No Observed Effect Concentration

50

Lowest Observed Effect Concentration

100

Reproduction

EC10

36

95% confidence limits

20 to 65

EC50

94

95% confidence limits

42 to >100

No Observed Effect Concentration

25

Lowest Observed Effect Concentration

50

Body Length

EC50

>100

95% confidence limits

Not determined

No Observed Effect Concentration

12.5

Lowest Observed Effect Concentration

25

Description of key information

A study was performed to assess the chronic toxicity of the test item to Daphnia magna.

Chemical analysis of the test preparations throughout the test period showed measured test concentrations of less than the LOQ of the analytical method employed were obtained which was determined to be 0.0010 mg/Ltitanium, equivalent to 0.0021 mg/L test item. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Exposure of Daphnia magna to the test item gave the following results based on the nominal test concentrations:

Endpoint

Concentration
(% v/v Saturated Solution)

Immobilization

EC50

>100

95% confidence limits

Not determined

No Observed Effect Concentration

50

Lowest Observed Effect Concentration

100

Reproduction

EC10

36

95% confidence limits

20 to 65

EC50

94

95% confidence limits

42 to >100

No Observed Effect Concentration

25

Lowest Observed Effect Concentration

50

Body Length

EC50

>100

95% confidence limits

Not determined

No Observed Effect Concentration

12.5

Lowest Observed Effect Concentration

25

In view of the difficulties associated with the evaluation of aquatic toxicity of the test item due to its poor water solubility in the daphnia reproduction study, a saturated solution was prepared by stirring an excess (100 mg/L) of test item in culture medium for a period of 24 hours. Any undissolved test item present was then removed by filtration (0.2 μm Sartorius Sartopore, first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated solution of the test item. A series of dilutions was made from this saturated solution to give further test concentrations of 6.25, 12.5, 25 and 50% v/v saturated solution

 

A sensitive method of analysis was developed for the daphnia reproduction study with a limit of quantitation (LoQ) of 0.0010 mg/L titanium, equivalent to 0.0021 mg/L test substance and this method was used to analysethe test preparations throughout the test period. However, despite the very low LoQ, no test substance was detected in any of the test solutions prepared.

 

As a result, the NOEC is reported as % v/v saturated solution used in the test. In the absence of any quantitative determination of the saturated solution concentration in either the water solubility or daphnia reproduction tests, it is not possible to  derive a quantitative value for the NOEC. Consequently, it is not possible to calculate a Predicted No Effect Concentration (PNEC). It is also not possible to estimate exposure for an unknown substance, since the physicochemical properties are required inputs for exposure estimation models. 

 

Furthermore, since the reported NOEC is neither quantitative nor supported by analytical data, it is proposed that the classification is assigned "inconclusive".

Key value for chemical safety assessment

Additional information

The reported NOEC is not supported by analytical data which showed test concentrations of less than the limit of quantitation (< 0.0010 mg/L titanium). The NOEC is given as v/v percentage of the saturated solution used in the test and in the absence of a quantitative value for the NOEC it is not possible to calculate a Predicted No Effect Concentration (PNEC). Similarly, it is not possible to estimate exposure or determine risk characterisation ratios for an unidentifed substance. Classification is inconclusive.