Reaction products of neodecanoic acid and titanium (IV) isopropoxide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 September 2009 and 6 November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

E0286P/040A

IUPAC Name:

E0286P/040A

Details on test material:

Sponsor's identification:E0286P/040A
Description :amber coloured viscous liquid
Batch number :E0289P/040A/01
Date received :20 July 2009
Storage conditions:room temperature on the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of test loading rates
Samples were taken from the control (replicates R1 - R6 pooled) and each loading rate WAF test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at each occasion and stored at approximately 20ºC for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in the attached Appendix 4.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test
Due to the low aqueous solubility and complex nature of the test material for the purposes of the test the test material was prepared as a Water Accommodated Fraction (WAF).
The loading rates to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Amounts of test material (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.
An aliquot (250 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (7.2 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test material.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.
Experimental Preparation
Amounts of test material (20, 40, 80, 160 and 320 mg) were each separately added to the surface of 2 litres of culture medium to give the 10, 20, 40, 80 and 160 mg/l loading rates respectively. After the addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10, 20, 40, 80 and 160 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.
An aliquot (500 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (4.4 ml) to give the required test concentrations of 10, 20, 40, 80 and 160 mg/l loading rate WAF.
The concentration and stability of neodecanoic acid in the test preparations were verified by chemical analysis at 0 and 72 hours (see Appendix 4).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test Species
The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°C.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 – 105 cells/ml.
A positive control (Harlan Laboratories Ltd Project Number: 0039/1088) used potassium dichromate as the reference material. The positive control was conducted between 26 May 2009 and 29 May 2009.
Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium is defined in Appendix 2 (see in any other materials and methods section).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded.

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.

pH:

The pH values of the control cultures (see Table 2 in any other information on results section) were observed to increase from pH 7.4 at 0 hours to 7.6 at 7.9 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth (see Figure 1 attached section) exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the EEC Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 250ml
- Aeration: No aeration

- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable

- Renewal rate of test solution (frequency/flow rate): Not applicable

- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.65 x 10E5 cells per ml. Inoculation of 1 litre of test medium with 11 ml of this algal suspension gave an initial nominal cell density of 4 x 10E3 cells per ml and had no significant dilution effect on the final test concentration.

- Control end cells density: algal cell density was approximately 6.24 10E5 cells/ml

- No. of organisms per vessel:
initial cell density of approximately 4 x 10E3 cells/ml.

- No. of vessels per concentration (replicates):
Three replicate flasks per concentration.

- No. of vessels per control (replicates):
Six replicate flasks.


GROWTH MEDIUM
- Standard medium used: yes
For the purpose of the definitive test, the test material was dissolved directly in culture medium. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).


TEST MEDIUM

Amounts of test material (20, 40, 80, 160 and 320 mg) were each separately added to the surface of 2 litres of culture medium to give the 10, 20, 40, 80 and 160 mg/l loading rates respectively. After the addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10, 20, 40, 80 and 160 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.
An aliquot (500 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (4.4 ml) to give the required test concentrations of 10, 20, 40, 80 and 160 mg/l loading rate WAF.




OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

- Light intensity and quality:
warm white lighting (380 – 730 nm)

EXPOSURE CONDITIONS :
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.55 x 105 cells per ml. Inoculation of 500 ml of test medium with 4.4 ml of this algal suspension gave an initial nominal cell density of 4 x 103 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.



EFFECT PARAMETERS MEASURED :
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). EL*x values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EL*50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
The results of the test are considered valid if the following performance criteria are met:
-The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
- The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
-The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL not stated

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 44 - 51 mg/l loading rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits 26 - 33 mg/l

Details on results:

Validation of Mixing Period
Pre-study work (see Appendix 3 in any other materials and methods section) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. Therefore it was considered appropriate to use a 24-Hour preparation period.
Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1 (see in any information on results section).
The results showed no effect on growth at 10 mg/l loading rate WAF. However, growth was observed to be reduced at 100 mg/l loading rate WAF.
Based on this information loading rates of 10, 20, 40, 80 and 160 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.
Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 (see in any information on results section). Daily specific growth rates for the control cultures are given in Table 3 (see in any information on results section). Growth rate and yield and values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 (see in any information on results section).
The mean cell densities versus time for the definitive test are presented in Figure 1 (see in attached section).
Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 38 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 4.50 x 103 cells per ml
Mean cell density of control at 72 hours : 1.73 x 105 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 32% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 6% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
Growth data
From the data given in Tables 2 and 4(see in any information on results section), it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period.
Accordingly the following results were determined from the data:
Inhibition of growth rate
ErL*10 (0 - 72 h) : 32 mg/l loading rate WAF
ErL*20 (0 - 72 h) : 37 mg/l loading rate WAF
ErL*50 (0 - 72 h) : 47 mg/l loading rate WAF; 95% confidence limits 44 - 51 mg/l loading rate WAF
where ErL*x is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 10 and 20 mg/l loading rate WAFs (P<0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 20 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 40 mg/l loading rate WAF.
Inhibition of yield
EyL*10 (0 - 72 h) : 17 mg/l loading rate WAF
EyL*20 (0 - 72 h) : 19 mg/l loading rate WAF
EyL*50 (0 - 72 h) : 29 mg/l loading rate WAF; 95% confidence limits 26 - 33 mg/l loading rate WAF
where EyL*x is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out. There were no statistically significant differences between the control, 10 and 20 mg/l loading rate WAFs (P<0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 20 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 40 mg/l loading rate WAF.
Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
Observations on test material solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period the 10, 20, 40 and 80 mg/l loading rate WAFs were observed to have formed clear colourless media columns with an oily scum of test material at the media surface whilst the 160 mg/l loading rate WAF was observed to have formed a clear colourless media column with an oily scum of test material at the media surface and globules of test material concentrated within the dimple. After the 1-Hour standing period the 10, 20, 40 and 80 mg/l loading rate WAFs were observed to have formed clear colourless media columns with an oily scum of test material at the media surface whilst the 160 mg/l loading rate WAF was observed to have formed a clear colourless media column with an oily scum and large globules of test material at the media surface. Microscopic examination of the WAFs showed there to be no micro-dispersions or globules of test material present.
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and 10 mg/l loading rate WAF test cultures were observed to be pale green dispersions. The 20 and 40 mg/l loading rate WAF test cultures were observed to be very pale green dispersions whilst the 80 and 160 mg/l loading rate WAF test cultures were observed to be clear colourless solutions.
Physico-chemical measurements
The pH values of each test and control flask are given in Table 2 (see in any information on results section) Temperature was maintained at 24 ± 1ºC throughout the test.
The pH values of the control cultures (see Table 2 in any other information on results section) were observed to increase from pH 7.4 at 0 hours to pH 7.6 – 7.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. The test material vessels showed a concentration dependant decline in pH at 0-hours with increasing test concentration in the range of pH 7.0 at 10 mg/l loading rate WAF through to pH 5.3 at 160 mg/l loading rate WAF. Whilst the inhibitory effect of the test material may be attributed to the concentration dependant decline in pH given that this was considered to be due to an intrinsic property of the test material no attempt was made to alter the pH prior to exposure.
Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface (see Table 5 in any other information on results section).
Chemical analysis of test loading rates
The test material is a mixture of titanium salts (80%) and alkyl acids (20%). Method development work conducted indicated that at the test concentrations to be employed in the definitive test analysis of the test samples for titanium by GC-MS was unreliable possibly due to interference from other components of the test material. Analysis by HPLC-MS did however give a good response 171 at m/z which corresponds to the neodecanoic acid present in the test material. At the request of the Sponsor analysis of the test samples for the presence of neodecanoic acid was performed to provide an indication of the presence of dissolved hydrocarbon constituents.
Analysis of the test preparations at 0 and 72 hours (see Appendix 4 in attached section) showed a concentration dependant increase in the measured concentrations of neodecanoic acid in the range of 6.8 mg/l at 10 mg/l loading rate WAF to 39 mg/l at 160 mg/l loading rate WAF.
Given that the toxicity of a complex UVCB chemical that has limited water solubility cannot be attributed to a single, or combination of, constituents the results are expressed based on nominal loading rates for the whole material.

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1088) used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure ofDesmodesmus subspicatus(CCAP 276/20) to the reference material gave the following results:

ErC50(0 – 72 h)        :          0.79 mg/l[1]
EyC50(0 – 72 h)       :          0.30 mg/l, 95% confidence limits 0.27 – 0.34 mg/l

No Observed Effect Concentration (NOEC) based on growth rate:              0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield:                   0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate:     0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield:                    0.50 mg/l The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

[1]It was not possible to calculate 95% confidence limits for the ErC50value as the data generated did not fit the models available for the calculation of confidence limits.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.15E+03	4.54E+05
	R2	4.40E+03	4.46E+05	-	-
	Mean	5.28E+03	4.50E+05
10	R1	6.17E+03	8.25E+05
	R2	3.67E+03	4.87E+05	[10]	[46]
	Mean	4.92E+03	6.56E+05
100	R1	6.02E+03	2.16E+04
	R2	5.62E+03	1.17E+04	76	98
	Mean	5.82E+03	1.67E+04

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

Table2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	47 h	72 h	72 h
Control	R1	7.4	4.26E+03	1.13E+04	2.86E+04	1.19E+05	7.9
	R2	7.4	4.35E+03	1.19E+04	2.79E+04	1.88E+05	7.9
	R3	7.4	4.60E+03	1.12E+04	2.97E+04	1.62E+05	7.8
	R4	7.4	4.26E+03	1.11E+04	2.94E+04	1.64E+05	7.7
	R5	7.4	4.42E+03	1.29E+04	2.86E+04	2.49E+05	7.7
	R6	7.4	5.12E+03	1.15E+04	2.95E+04	1.53E+05	7.6
	Mean		4.50E+03	1.17E+04	2.89E+04	1.73E+05
10	R1	7.0	4.85E+03	1.01E+04	2.88E+04	2.22E+05	7.6
	R2	7.0	4.43E+03	8.68E+03	2.70E+04	2.77E+05	7.6
	R3	7.0	4.66E+03	1.11E+04	3.54E+04	1.88E+05	7.6
	Mean		4.65E+03	9.95E+03	3.04E+04	2.29E+05
20	R1	6.7	4.52E+03	1.13E+04	2.38E+04	1.73E+05	7.5
	R2	6.7	4.33E+03	8.97E+03	1.91E+04	1.13E+05	7.5
	R3	6.7	4.87E+03	9.98E+03	2.47E+04	1.07E+05	7.5
	Mean		4.57E+03	1.01E+04	2.25E+04	1.31E+05
40	R1	6.5	4.46E+03	7.12E+03	2.11E+04	1.07E+05	7.4
	R2	6.5	4.33E+03	6.67E+03	2.12E+04	3.61E+04	7.4
	R3	6.5	4.33E+03	6.06E+03	2.15E+04	5.86E+04	7.3
	Mean		4.37E+03	6.62E+03	2.13E+04	6.71E+04
80	R1	5.9	4.54E+03	2.89E+03	4.34E+03	5.38E+03	6.9
	R2	5.9	4.27E+03	2.34E+03	3.82E+03	4.18E+03	6.9
	R3	5.9	4.18E+03	2.73E+03	3.77E+03	4.34E+03	6.7
	Mean		4.33E+03	2.65E+03	3.98E+03	4.63E+03
160	R1	5.3	4.71E+03	1.43E+03	3.21E+03	2.16E+03	6.0
	R2	5.3	4.50E+03	2.18E+03	1.18E+03	2.81E+03	5.8
	R3	5.3	4.30E+03	1.98E+03	7.92E+02	2.37E+03	5.8
	Mean		4.50E+03	1.86E+03	1.73E+03	2.45E+03

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.043	0.040	0.057
	R2	0.045	0.037	0.076
	R3	0.043	0.042	0.068
	R4	0.043	0.042	0.069
	R5	0.049	0.035	0.087
	R6	0.044	0.041	0.066
	Mean	0.045	0.040	0.071

R₁- R₆= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield and in the Definitive Test

Nominal Loading Rate (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.047		1.15E+05
	R2	0.053		1.84E+05
	R3	0.051		1.58E+05
	R4	0.052	-	1.59E+05	-
	R5	0.057		2.45E+05
	R6	0.051		1.48E+05
	Mean	0.052		1.68E+05
	SD	0.003		4.38E+04
10	R1	0.056	[8]	2.17E+05
	R2	0.059	[13]	2.73E+05
	R3	0.053	[2]	1.83E+05
	Mean	0.056	[8]	2.24E+05	[34]
	SD	0.003		4.53E+04
20	R1	0.052	0	1.69E+05
	R2	0.046	12	1.08E+05
	R3	0.046	12	1.02E+05
	Mean	0.048	8	1.27E+05	25
	SD	0.003		3.66E+04
40	R1	0.046	12	1.02E+05
	R2	0.031	40	3.18E+04
	R3	0.037	29	5.43E+04
	Mean	0.038	27	6.28E+04	63
	SD	0.008		3.59E+04
80	R1	0.004	92	8.32E+02
	R2	0.001	98	-9.60E+01
	R3	0.001	98	1.52E+02
	Mean	0.002	96	2.96E+02	100
	SD	0.002		4.80E+02
160	R1	-0.009	117	-2.55E+03
	R2	-0.005	110	-1.69E+03
	R3	-0.007	113	-1.93E+03
	Mean	-0.007	113	-2.06E+03	101
	SD	0.002		4.46E+02

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		10		20
	*	+	*	+	*	+
Height of Media Column (cm)	12	12	12	12	12	12
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

	Nominal Loading Rate (mg/l)
	40		80		160
	*	+	*	+	*	+
Height of Media Column (cm)	12	12	12	12	12	12
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

*= Start of mixing period

+= End of mixing period

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSION
The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results based on nominal loading rates:
Response Variable EL*50 95% Confidence Limits No Observed Effect Loading Rate Lowest Observed Effect Loading Rate
(mg/l Loading Rate WAF) (mg/l Loading Rate WAF) (NOEL) (mg/l) (LOEL) (mg/l)
Growth Rate 47 44 - 51 20 40
Yield 29 26 - 33 20 40

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods. 

Information provided by the Sponsor indicated that the test material was a mixture of titanium salts and alkyl acids. Given this and also the poor solubility of the test material in water the most appropriate method of preparation for this material was as a Water Accommodated Fraction (WAF). 

Following a preliminary range-finding test Desmodesmus subspicatus was exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 20, 40, 80 and 160 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results.

Exposure of Desmodesmus subspicatus to the test material gave the following results based on nominal loading rates:

Response Variable	EL*50 (mg/l Loading Rate WAF)	95% Confidence Limits (mg/l Loading Rate WAF)			No Observed Effect Loading Rate (NOEL) (mg/l)	Lowest Observed Effect Loading Rate (LOEL) (mg/l)
Growth Rate	47	44	-	51	20	40
Yield	29	26	-	33	20	40

The test material is a mixture of titanium salts (80 %) and alkyl acids (20%). Method development work conducted indicated that at the test concentrations to be employed in the definitive test analysis of the test samples for titanium by GC-MS was unreliable possibly due to interference from other components of the test material. Analysis by HPLC-MS did however give a good response at 171 m/z which corresponds to the neodecanoic acid present in the test material. At the request of the Sponsor analysis of the test samples for the presence of neodecanoic acid was performed to provide an indication of the dissolved hydrocarbon content.

Analysis of the test preparations at 0 and 72 hours showed a concentration dependant increase in the measured concentrations of neodecanoic acid in the range of 6.8 mg/l at 10 mg/l loading rate WAF to 39 mg/l at 160 mg/l loading rate WAF.

Given that the toxicity of a complex UVCB chemical that has limited water solubility cannot be attributed to a single, or combination of, constituents the results are expressed based on nominal loading rates for the whole material.

*EL = Effective Loading Rate

CONCLUSION

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results based on nominal loading rates:

Response Variable	EL*50 (mg/l Loading Rate WAF)	95% Confidence Limits (mg/l Loading Rate WAF)			No Observed Effect Loading Rate (NOEL) (mg/l)	Lowest Observed Effect Loading Rate (LOEL) (mg/l)
Growth Rate	47	44	-	51	20	40
Yield	29	26	-	33	20	40

*EL = Effective Loading Rate