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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
10 September 2012- 08 November 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study performed according to OECD guideline 201 (2006). category and read-across justification: The substance is hydrolytically unstable. When it comes in contact with water or moisture, a complete hydrolysis will take place with no significant reaction products other than the particular alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the fate and pathways of the substance. The aquatic toxicity testing is considered scientifically unjustified as the substance degrades immediately releasing the particular alcohol and hydrated insoluble titanium dioxides in water. The testing conducted with analogue substance of the category justifies that the aquatic toxicity in daphnia and algae studies is similar to the aquatic toxicity of the alcohol released to test water as the insoluble hydrated titanium oxide, precipitated on the bottom of the test vessels is lacking bioavailability. The identification of the degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance which might change the aquatic toxicity of the target substance compared to the toxicity of the pure alcohol. As a conclusion, the read-across approach from analogue category member is used to justify that the testing is scientifically unjustified for the target substance and the read-across from the degradation product (relevant alcohol) can be used to evaluate the aquatic toxicity and the fate and pathways of the target substance.
Justification for type of information:
The substance is hydrolytically unstable. When it comes in contact with water or moisture, a complete hydrolysis will take place with no significant reaction products other than the particular alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the fate and pathways of the substance. The aquatic toxicity testing is considered scientifically unjustified as the substance degrades immediately releasing the particular alcohol and hydrated insoluble titanium dioxides in water.
The testing conducted with analogue substance of the category justifies that the aquatic toxicity in daphnia and algae studies is similar to the aquatic toxicity of the alcohol released to test water as the insoluble hydrated titanium oxide, precipitated on the bottom of the test vessels; lacking bioavailability. The identification of the degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance which might change the aquatic toxicity of the target substance compared to the toxicity of the pure alcohol.
As there is a mechanistic reasoning to the read-across, the read-across from the degradation product (relevant alcohol) is used to evaluate the aquatic toxicity and the fate and pathways of the target substance in the environment.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: solutions containing 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 % of the filtrate prepared at 100 mg test item per litre.
- Sample storage conditions before analysis: Samples were stored in freezer untill analysis

Duplicate samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h, t=24 h and t=72 h
Volume 1 ml
Storage Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period. 1 to 2 replicates were taken of each test concentration for sampling purposes.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Eluate: Dilution water
- Differential loading: Solutions containing 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 % of the filtrate prepared at 100 mg test item per litre. As the test item is hydrolytically unstable concentrations of the hydrolysis products being octyl alcohol isomers were measured. At the start of the test, the mean measured concentrations were 1.96, 7.0, 19 and 57 mg/l in test groups representing 3.2, 10, 32 and 100% of the filtrate, respectively. The mean measured concentrations were at the level of 45 to 95 % of initial after 24 hours of exposure. At the end of the test the actual concentrations were below 0.7 mg/l at 3.2 and 10% of the filtrate, below 1.4 mg/l at 32% of the filtrate and below 2.8 mg/l in the undiluted filtrate.

The measured concentration in the samples taken from the undiluted filtrate incubated without algae followed similar pattern.
Note that the concentrations should be considered indicative.
Based on these results, the Time Weight Average concentrations were calculated.

-Replicates: 3 replicates of each concentration, 6 replicate of the control, 1 replicate of each concentration without algae, and 1 to 2 replicates of each test concentration for sampling purposes.


- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Preparation of test solutions started with a loading rate of 100 mg/l applying a 15 to 17-minute period of magnetic stirring that was sufficient to ensure complete hydrolysis to octyl alcohol isomers. The obtained clear and colourless solution containing precipitate and a floating layer of undissolved material was filtered through a 0.45 µm membrane filter (rc55, Whatman) and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture (NOTOX)
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use. An initial cell density of 1 x 104 cells/ml was used in the test.
- Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Any deformed or abnormal cells observed: No abnormal cells observed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
0-72 h
Hardness:
(Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
The temperature of the test medium was 21.5°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.0 and 23.2°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).
pH:
The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit)
Nominal and measured concentrations:
Concentrations of the hydrolysis products being octyl alcohol isomers were measured. At the start of the test, the mean measured concentrations were 1.96, 7.0, 19 and 57 mg/l in test groups representing 3.2, 10, 32 and 100 % of the filtrate, respectively. The mean measured concentrations were at the level of 45 to 95% of initial after 24 hours of exposure. At the end of the test the actual concentrations were below 0.7 mg/l at 3.2 and10 % of the filtrate, below 1.4 mg/l at 32% of the filtrate and below 2.8 mg/l in the undiluted filtrate (note that the concentrations should be considered indicative). The Time Weight Average concentrations were calculated 0.81, 3.3, 6.7 and 20 mg octyl alcohol isomers per litre in in test groups representing 3.2, 10, 32 and 100 % of the filtrate, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Type (delete if not applicable): Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: An initial cell density of 1 x 10^4 cells/ml
- Control end cells density: 113.6 x 10^4 cells/ml

GROWTH MEDIUM
- Standard medium used: yes
-Stock culture medium: According to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/l
K2HPO4.3H2O 52 mg/l
MgSO4.7H2O 75 mg/l
Na2CO3.10H2O 54 mg/l
C6H8O7.H2O 6 mg/l
NH4NO3 330 mg/l
CaCl2.2H2O 35 mg/l
C6H5FeO7.xH2O 6 mg/l
H3BO3 2.9 mg/l
MnCl2.4H2O 1.81 mg/l
ZnCl2 0.11 mg/l
CuSO4.5H2O 0.08 mg/l
NH4)6Mo7O24.4H2O 0.018 mg/l

TEST MEDIUM / WATER PARAMETERS
Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 50 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
pH 8.1 ± 0.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 88 to
89 E.m-2.s-1.
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.
Observations/measurements in the study were recorded electronically using the following programme(s):
-Shimadzu Spectrophotometer UV-1800 including UVProbe 2.33 software (Shimadzu, Kyoto, Japan): Algal cell density.
-REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature.
-Appearance of the cells : At the end of the final test microscopic observations were performed at the two highest concentrations to observe for any abnormal appearance of the algae
-pH: At the beginning and at the end of the test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test.
-Temperature of medium: Continuously in a temperature control vessel.

TEST CONCENTRATIONS
-A combined limit/range-finding test: Six replicates of exponentially growing algae were exposed to a control and an undiluted filtrate prepared at 100 mg/l. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates per concentration were exposed to 0.1, 1.0 and 10% of the filtrate.
• pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed to verify a normal and healthy appearance.
• Singular samples for possible analysis were taken in the combined limit/range-finding test.

Calibration curve
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period. 

Comparison of average growth rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments: 

µi-j = lnXj-lnXi/tj-ti (day-1)

Where:  
 µi-j = the average specific growth rate from time i to j              
Xi= the biomass at time i              
Xj= the biomass at time j
The average growth rate at each test substance concentration is then compared with the control value and the percentage reduction in growth rate is calculated: 

%lr=µc-µt/µc*100  
Where:   
%Ir = percent inhibition in average specific growth rate            
 µC= mean value for average specific growth rate in the control group             
 µT= average specific growth rate for the treatment replicate  

The percent inhibition in yield is calculated for each treatment replicate as follows:  

%ly=Yc-Yt/Yc×100   

Where:   %Iy = percent inhibition of yield            
YC= mean value for yield in the control group              
YT= value for yield for the treatment replicate

Determination of the average exposure concentrations
The average exposure concentrations were calculated as: 24×√Ct=0×Ct=24+48×√Ct=24×Ct=72 /72  ,being the Time Weight Average () of the concentrations of octyl alcohol isomers measured in the samples taken at the start (Ct=0), after 24 hours (Ct=24) and the end of the test (Ct=72).   In case concentrations measured are below the limit of detection or the limit of quantification, the final exposure concentration(s) will be taken as a factor of 2 below the applicable limit. This procedure is based on the OECD “Guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment”.

- Test concentrations:
Six exponentially growing algal cultures were exposed to a control whereas three replicates per group were exposed to solutions containing 0.1, 0.32, 1.0, 3.2, 10, 32 and 100% of the filtrate prepared at 100 mg/l. The initial cell density was 104 cells/ml and the total exposure period was 72 hours. Samples for analytical confirmation of the exposure concentrations were taken at the start, 24 hours after start of the test and at the end of the exposure.

Concentrations of the hydrolysis products being octyl alcohol isomers were measured. At the start of the test, the mean measured concentrations were 1.96, 7.0, 19 and 57 mg/l in test groups representing 3.2, 10, 32 and 100% of the filtrate, respectively. The mean measured concentrations were at the level of 45 to 95% of initial after 24 hours of exposure. At the end of the test the actual concentrations were below 0.7 mg/l at 3.2 and10% of the filtrate, below 1.4 mg/l at 32% of the filtrate and below 2.8 mg/l in the undiluted filtrate (note that the concentrations should be considered indicative). The Time Weight Average concentrations were calculated 0.81, 3.3, 6.7 and 20 mg octyl alcohol isomers per litre in in test groups representing 3.2, 10, 32 and 100% of the filtrate, respectively

- Results used to determine the conditions for the definitive study:
A final test was performed based on the results of a combined limit/range-finding test. Preparation of test solutions started with a loading rate of 100 mg/l applying a 15 to 17-minute period of magnetic stirring that was sufficient to ensure complete hydrolysis to octyl alcohol isomers. The obtained clear and colourless solution containing precipitate and a floating layer of undissolved material was filtered through a 0.45 µm membrane filter and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.


Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 6.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: octyl alcohol isomers
Basis for effect:
growth rate
Remarks:
Represents 32 % of a filtrate prepared at 100 mg Titanium tetra(octanolate), branched and linear per litre.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 32 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Represents measured octyl alcohol isomers concentration of 6.7 mg/l.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 20 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: octyl alcohol isomers
Basis for effect:
growth rate
Remarks:
EC50 was beyond the range tested. TWA conc. represents conc. of 100 % undiluted filtrate prepared at 100 mg test item per litre.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
EC50 was beyond the range tested. Represents measured conc. of octyl alcohol isomers of 20 mg/l.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: octyl alcohol isomers
Basis for effect:
biomass
Remarks:
Represents 10 % of a filtrate prepared at 100 mg test item per litre.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 10 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Represents the measured octyl alcohol isomers conc. of 3.3 mg/l.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 10 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: octyl alcohol isomers
Basis for effect:
biomass
Remarks:
Undiluted test item inhibited the yield of this fresh water algae species significantly at a TWA concentration of 6.7 mg octyl alcohol isomers per litre.
Remarks on result:
other: 2.2 to 46 mg/l
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control
- Unusual cell shape:not observed
- Colour differences:not observed
- Flocculation:not observed
- Adherence to test vessels:not observed
- Aggregation of algal cells:not observed
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
The sponsor supplied information that the test substance is highly hydrolytically unstable. Therefore, the effect parameters were based on the concentration of the hydrolysis product of the test substance, i.e. octyl alcohol isomers (Alcohols, C7-9-ISO, C8-rich).The batch of Titanium tetra(octanolate), branched and linear tested was a clear colourless to pale yellow liquid and a UVCB substance. Because of high water reactivity of the test item preparation of test solutions started with a loading rate of 100 mg/l applying a 15 to 17-minute period of magnetic stirring that was sufficient to ensure complete hydrolysis to octyl alcohol isomers. The obtained clear and colourless solution containing a white precipitate (hydrated titanium dioxide) and a floating layer of undissolved material. This precipicate was filtered through a 0.45 µm membrane filter (rc55, Whatman) and the solution used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.



Results with reference substance (positive control):
- Results with reference substance: valid
Pseudokirchneriella subcapitata, strain: NIVA CHL-1. Fresh water algal growth inhibition test with potassium dichromate (Project 501711).
Start of first exposure: 20 November 2012
Completion last exposure: 23 November 2012
Potassium dichromate (Merck, Art. 1.04864, Batch K34869764 607).
Algae were exposed for a period of 72 hours to concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a control. The initial cell density was 1.0 x 104 cells/ml.

- EC50:
Potassium dichromate reduced growth rate of this fresh water algae species biologically significantly (>10%) at nominally 1.0 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.7 mg/l with a 95% confidence interval ranging from 1.2 to 2.3 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.78 mg/l with a 95% confidence interval ranging from 0.60 to 1.0 mg/l. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:
Calculation of the EC50and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the octyl alcohol isomers.

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic ToxicologyGuidelines" by S. Pack, August 1993.

Table 1    Mean cell densities (x104cells/ml) during the combined limit/range-finding test

Test substance1

% of filtrate

prep. at 100 mg/l 

Exposure time (hours)

 

 

 

 

0

24

48

72

control

1.0

3.5

12.7

46.7

0.10

1.0

3.3

12.3

48.9

1.0

1.0

3.3

11.2

42.1

10

1.0

4.1

12.2

37.5

100

1.0

6.2

7.8

4.1

1.Titanium tetra(octanolate), branched and linear

 

Table 2      Percentage reduction of growth rate and inhibition of yield during the combined limit/range-finding test

Test substance1

% of filtrate

prep. at 100 mg/l

Mean growth rate

Yield (0-72 h)

 

 

 

 

µ (0-72 h)

Reduction (%)

x104cells/ml

Inhibition (%)

control

0.05334

 

45.71

 

0.10

0.05400

-1.2

47.92

-4.8

1.0

0.05131

3.8

41.06

10.2

10

0.05029

5.7

36.45

20.3

100

0.01970

63.1

3.15

93.1

1.Titanium tetra(octanolate), branched and linear

Table 3            Measured concentrations versus nominal concentrations

Test substance1

% of filtrate

prep. at 100 mg/l

Measured concentration (mg/l)

t=0h

t=24h

t=72h

TWA

Mean

Mean

Mean2

(mg/l)

3.2

1.88

1.96

0.906

0.87

< 0.7

0.35

0.81

3.2

2.04

0.843

< 0.7

10

5.87

7.0

5.86

6.7

< 0.7

0.35

3.3

10

8.14

7.5

< 0.7

32

18.5

19

11

11

< 1.4

0.7

6.7

32

19.7

10.9

< 1.4

100

57.3

57

35.3

36

< 2.8

1.4

20

100

57.4

36.8

< 2.8

1.   Titanium tetra(octanolate), branched and linear

Half of the lowest calibration standard taking dilution factor into account

Table 4        Mean cell densities (x 104cells/ml) during the final test

Test substance1

Exposure time (hours)

% of filtrate

 

 

 

 

prep. at 100 mg/l

0

24

48

72

control

1.0

5.8

27.1

113.6

0.10

1.0

6.0

27.6

117.5

0.32

1.0

6.3

29.6

126.2

1.0

1.0

5.7

24.1

103.1

3.2 (0.81)

1.0

6.0

28.9

118.6

10 (3.3)

1.0

5.5

25.0

106.3

32 (6.7)

1.0

5.4

20.6

87.5

100 (20)

1.0

4.3

8.3

18.0

1. Titanium tetra(octanolate), branched and linear

() – TWA concentration of octyl alcohol isomers (mg/l) Table 5 Percentage reduction of growth rate (total test period) and percentage inhibitionof yield during the final test

Test substance1

% of filtrate

prep. at 100 mg/l

Mean growth rate

Yield (0-72 h)

 

 

 

 

µ (0-72 h)

Reduction (%)

x104cells/ml

Inhibition (%)

control

0.06570

 

112.64

 

0.10

0.06617

-0.7

116.51

-3.4

0.32

0.06717

-2.2

125.15

-11.1

1.0

0.06431

2.1

102.12

9.3

3.2 (0.81)

0.06630

-0.9

117.59

-4.4

10 (3.3)

0.06478

1.4

105.29

6.5

32 (6.7)

0.06210

5.5

86.47

23.2*

100 (20)

0.03948

39.9*

16.98

84.9*

1. Titanium tetra(octanolate), branched and linear

() – TWA concentration of octyl alcohol isomers (mg/l)

* - statistically significant effect (p<0.05)

 

Table 6            Percentage reduction of growth rate at different time intervals during the final test

Test substance1

% of filtrate

prep. at 100 mg/l

Mean growth rate

 

 

 

 

 

 

µ (0-24 h)

Reduction (%)

µ (24-48 h)

Reduction (%)

µ (48-72 h)

Reduction (%)

control

0.07295

 

0.06446

 

0.05968

 

0.10

0.07453

-2.2

0.06371

1.2

0.06027

-1.0

0.32

0.07654

-4.9

0.06460

-0.2

0.06037

-1.2

1.0

0.07236

0.8

0.05995

7.0

0.06063

-1.6

3.2 (0.81)

0.07455

-2.2

0.06560

-1.8

0.05876

1.5

10 (3.3)

0.07068

3.1

0.06324

1.9

0.06043

-1.2

32 (6.7)

0.07023

3.7

0.05576

13.5

0.06031

-1.1

100 (20)

0.06040

17.2

0.02787

56.8

0.03017

49.4

1. Titanium tetra(octanolate), branched and linear

() – TWA concentration of octyl alcohol isomers (mg/l)

Validity criteria fulfilled:
yes
Remarks:
In the control, cell density increased by an average factor of >16 within 2 days. Section-by-section specific growth rates in the control cultures did not exceed 35%. Specific growth rates in replicate control cultures did not exceed 7%.
Conclusions:
The 72-h EC50 (growth rate) to Pseudokirchnerella subcapitata was measured using OECD 201 in accordance with GLP. As the test substance hydrolyses during testing releasing alcohol and hydrated titanium dioxide in water, the test concentrations were measured based on the released octyl alcohol isomers. Test substance reduced growth rate at a TWA concentration of 20 mg octyl alcohol isomers per litre and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 6.7 mg octyl alcohol isomers per litre.

The EC50 for growth rate reduction (ERC50: 0-72h) was beyond the range tested, i.e. exceeded a TWA concentration of 20 mg octyl alcohol isomers per litre obtained in the undiluted filtrate prepared at 100 mg Titanium tetra(octanolate), branched and linear per litre.

The EC50 for yield inhibition (EYC50: 0-72h) was 10 mg/l with a 95 % confidence interval ranging from 2.2 to 46 mg/l.

The NOEC for growth rate reduction was 6.7 mg/l based on a TWA concentration of octyl alcohol isomers, which represents 32 % of a filtrate prepared at 100 mg Titanium tetra(octanolate), branched and linear per litre. The NOEC for yield inhibition was 3.3 mg/l based on a TWA concentration of octyl alcohol isomers which represents 10 % of a filtrate prepared at 100 mg Titanium tetra(octanolate), branched and linear per litre.
Executive summary:

This read-across substance is an analogue substance of the target substance (titanium tetrakis(2 -ethylhexanolate) in the category of highly water reactive titanates. It contains commercial solvent (alcohols C7 -9-iso, C8-rich, CAS no 68526 -83 -0). This solvent consists mainly of isooctanol (93 %), which is similar to the 2 -ethylhexanol released from the target substance. This data is used as a weight of evidence to justify that all the organometallic titanates grouped into this category hydrolyse during toxicity testing, and therefore the intrinsic properties are related to the toxicity of the alcohol released from the substance.

The acute toxicity test performed for this substance identifies that the intrinsic properties of this substance are related to the degradation products due to the rapid hydrolysis which takes place immediately after test substance is introduced to water solution.

This toxicity study is classified as reliable and satisfies the guideline requirements for the acute invertebrate toxicity study. It is used as part of a weight of evidence to evaluate the toxicity of the target substance titanium tetrakis(2 -ethylhexanolate).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
19.3.1991 - 06.09.1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to GLP guideline study. Read-across justification: The substance is hydrolytically unstable. When it comes in contact with water or moisture complete hydrolysis will take place with no significant reaction products other than the particular alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the fate and pathways of the substance. The aquatic toxicity testing is considered scientifically unjustified as the substance degrades immediately releasing alcohol and hydrated insoluble titanium dioxides in water. The testing conducted with analogue substances of the category proves that the aquatic toxicity in daphnia and algae studies is similar to the aquatic toxicity of alcohol released to the test water, and the insoluble hydrated titanium oxides precipitated on the bottom of the test vessels. The identification of degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance, which might change the aquatic toxicity of the target substance compared to the toxicity of the pure alcohol. As there is a mechanistic reasoning to the read-across, the read-across from the degradation product (relevant alcohol) is used to evaluate the aquatic toxicity and the fate and pathways of the target substance in the environment.
Justification for type of information:
The substance is hydrolytically unstable. When it comes in contact with water or moisture, a complete hydrolysis will take place with no significant reaction products other than the particular alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the fate and pathways of the substance. The aquatic toxicity testing is considered scientifically unjustified as the substance degrades immediately releasing the particular alcohol and hydrated insoluble titanium dioxides in water.
The testing conducted with analogue substance of the category justifies that the aquatic toxicity in daphnia and algae studies is similar to the aquatic toxicity of the alcohol released to test water as the insoluble hydrated titanium oxide, precipitated on the bottom of the test vessels; lacking bioavailability. The identification of the degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance which might change the aquatic toxicity of the target substance compared to the toxicity of the pure alcohol.
As there is a mechanistic reasoning to the read-across, the read-across from the degradation product (relevant alcohol) is used to evaluate the aquatic toxicity and the fate and pathways of the target substance in the environment.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
EEC 88/302 C.3
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of stock solution (500 mg/L) using de-ionised water
- Controls: negative control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: (CHODAT (86.81 SAG))
- Source (laboratory, culture collection): Institut für Wasser-, Boden- und Lufthygiene, Berlin; followed by in-house breeding.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: a pre-culture was grown from the stock 3 days before initiation. Initiation occurred at a density of 20,000 cells/mL.


ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
according to guideline EEC 88/302
Test temperature:
22°C
pH:
8.1 -8.3 at initiation
Nominal and measured concentrations:
nominal: 0, 1, 2, 4, 8, 16, 32 mg/L
Measured: no
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: sterile Erlenmeyer flasks
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 20,000/mL
- Control end cells density: 820,000
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: de-ionised water


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 8000 lux, white


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer, wave lenth 685 nm; examinations at 0, 24, 48, and 72 h after initiation
- Chlorophyll measurement: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Mean cell counts (x E04 cells/mL)

Test concentration

[mg/L]

Time [h]

0

24

48

72

0

2

7

25

82

1

2

7

24

80

2

2

7

25

79

4

2

6

22

71

8

2

6

20

52

16

2

6

15

22

32

2

4

5

6

Read-across justifications and data matrices are presented in IUCLID section 13.

Conclusions:
The effect of 2 -ethylhexanol on algal growth was tested in a GLP guideline study (EEC 88/302 C.3). The EC50 (72 h) to Scenedesmus subspicatus was 11.5 mg/L based on biomass, and 16.6 mg/L based on the growth rate. The NOEC was 2.0 mg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Adequacy of study:
other information
Justification for data waiving:
other:

Description of key information

Testing is not scientifically justified as this substance is hydrolytically unstable. Therefore, the results (72-h EC50 and NOEC) of 2-ethylhexanol is used as a key value.

Key value for chemical safety assessment

EC50 for freshwater algae:
16.6 mg/L
EC10 or NOEC for freshwater algae:
2 mg/L

Additional information

When this substance comes in contact with water or moisture, a complete hydrolysis will take place with no significant reaction products other than EHD and hydrated titanium dioxide (half-life < 10 minutes, Brekelmans M.J.C. 2013). EHD and the structurally similair hydrolysis product, 2 -ethylhexanol, is readily biodegradable and not persistent (log Kow value 2.9 < 4). Therefore, the testing was considered unnecessary. The CSA indicates that toxicity of this substance is similar to 2 -ethylhexanol based on the weight of evidence.

Key studies (short-term toxicity to daphnia and algae) conducted for the analogue category member (titanium tetra(octanolate), branched and linear, CAS 68526 -83 -0) justify that these organometallic titanates hydrolyse during toxicity testing and the aquatic toxicity is similar to the alcohol released in water. Category and read-across justifications are presented in the annexes of the CSR.

This read-across substance is an analogue substance grouped together with the target substance into the category of highly water reactive titanates. It contains commercial solvent (Alcohols C7 -9-iso, C8-rich, CAS no 68526 -83 -0). This solvent contains 93 % of isooctanol being also an analogue substance with 2-ethylhexanol; an isomer of octyl alcohol.

The 72-h EC50 (growth rate) to Pseudokirchnerella subcapitata measured by using OECD 201 in accordance with GLP was conducted for the analogue category member. As the test substance hydrolysed during testing releasing alcohol and hydrated titanium dioxide in water, the test concentrations were measured based on the released octyl alcohol isomers. Test substance reduced growth rate at a TWA concentration of 20 mg octyl alcohol isomers per litre. The EC50 for growth rate reduction (ERC50: 0-72h) was beyond the range tested. The NOEC for growth rate reduction was 6.7 mg/l based on a TWA concentration of octyl alcohol isomers, which represents 32 % of a filtrate prepared at 100 mg titanium tetra(octanolate), branched and linear per litre.

In addition, a reliable 72-h EC50 (growth rate) (72 h) of 2-ethylhexanol to Scenedesmus subspicatus (new name: Desmodesmus subspicatus) is 16.6 mg/L, and the NOEC is 2.0 mg/L. This study test was conducted according EEC 84/449, C.2 ( i.e. similar to OECD test guideline 201) in accordance with GLP (Scholz 1991).

Based on the weight of evidence approach, the results of the analogue category member and the degradation product are relatively similar. The key NOEC and EC50-values for CSA are obtained from the lowest values i.e. the values of 2 -ethylhexanol.