Fatty acids, lanolin, lithium salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is GLP compliant and follows OECD guideline 201. Therefore, it has been given a reliability score of 1.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

See principles of method if other than guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

See principles of method if other than guideline

Principles of method if other than guideline:

Full chemical analysis was not conducted in the range finding test but as the results are based on a final definitive test with full supporting analysis this was not considered to affect the validity of the test. In the final test, cells from stock culture were used for inoculation of test solutions because the pre-culture showed insufficient growth. The validity criteria were met in the final study and study integrity was not adversely affected.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

The confimatory chemical analysis was based on the concentration of the fatty acid component (sebacate) and on the concentration of the lithium ion.Samples for analysis were taken from all test concentrations at the start, after 24 and 72 hours of exposure (lithium and sebacate analysis) and at the test start and after 72 hours of exposure (TOC analysis). Samples were stored in a refrigerator (TOC) or freezer (lithium and sebacate) 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 an intermediate 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.
For the determination of the test substance based on sebacate, the samples were diluted in a 1:1 (v:v) ratio with 2% formic acid in acetonitrile and analysed. If necessary, the samples were further diluted with 1% formic acid in 50/50 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range.
For the determination of the test substance based on lithium the samples were diluted in a 24:1 (v:v) ratio with HNO3 and analysed. If necessary, the samples were further diluted with 4% HNO3 in M2-medium to obtain concentrations within the calibration range.

Vehicle:

no

Details on test solutions:

Nominal concentrations 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg dilithium sebacate/L.
Medium: M2
Solubility: The test item was completely soluble in test medium at the concentrations tested.
Preparation of test solutions: A solution with a concentration of 100 mg/L was prepared by 20-50 minutes magnetic stirring and 6-8 minutes ultrasonic treatment. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
Appearance: The final test solutions were all clear and colourless.

Test organisms (species):

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

Details on test organisms:

Strain: NIVA CHL 1 from an in-house laboratory culture
Final test inoculation: Cells taken from stock culture
Stock culture: M1 growth medium inoculated with algal cells from pure culture on agar. Suspensions were continuously aerated and exposed to light at 21-24°C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

24 mg CaCO3/L

Test temperature:

22-24°C

pH:

7.9-8.2

Dissolved oxygen:

No data reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg Dilithium sebacate/L. For measured concentrations, please see additional information box.
The fact that the TOC concentrations and the concentration measured in the solution incubated without algae (3.2 mg/L) were stable during the exposure indicates that sebacate was absorbed by the algal biomass rather than degraded. This means that the algae were exposed to sebacate during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium sebacate. To cover the worst-case-scenario, effect parameters based on TWA concentrations were also calculated.

Details on test conditions:

Controls: Blank (test medium without test substance)
Solutions: 50 mL test solutions with 1 mL algae suspension (cell density of 10 (4) cells/mL)
Replicates: 3 per test concentration, 6 for control, 1 per test concentration for 24 hour sampling, 4 per group without algae for TOC analysis
Test vessels: 100 mL, all glass, capped
Illumination: Continuous
Incubation: Vessels were randomly distributed in the incubator and daily repositioned.
Agitation: Continuous shaking.
Observations: At the beginning of the test, cells were counted using a microscope and counting chamber and thereafter, cell densities were determined by spectrographic measurement, with algal medium as the blank. Microscopic observations were performed at the two highest test concentrations at the end of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Lower 95% CL: 7.5 mg/L. Upper 95% CL: 13 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Lower 95% CL: 1.6 mg/L. Upper 95% CL: 5.2 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Lower 95% CL: 17 mg/L. Upper 95% CL: 28 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Growth rates were in the range of the controls at the concentrations from 0.10 to 3.2 mg/L during the 72-hour test period. Statistically significant inhibition of growth rate was found at test concentrations of 10 mg/L and higher. Although a significant effect was determines at 10 mg/L dilithiumsebacate the observed effect was less than 10% of the control and was considered to be biologically insignificant. The NOEC for growth rate inhibition was therefore considered to be 10 mg/L dilithium sebacate.
Inhibition of growth rate decreased as exposure progressed.
Statistically significant inhibition of yield was found at test concentrations of 3.2 mg/L and higher so the NOEC for yield inhibition was set at 1.0 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher. The growth rate 72h-EC50 was 1.3 mg/L (95% confidence interval: 1.2 to 1.5 mg/L). The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L, so result corresponds with this range. The yield inhibition 72h-EC50 was 0.46 mg/L (95% confidence interval: 0.43 to 0.49 mg/L). The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L so result was within the low end of this range.

Reported statistics and error estimates:

Quantification of cell densities was based on a calibration curve. The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass, which is compared with the control value and the percentage inhibition in growth rate is calculated.
In case concentrations measured were below the limit of detection or the limit of quantification, the final exposure concentration(s) were taken as a factor of 2 below the applicable limit.
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 inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test substance.
The calculations were performed with ToxRat Professional v. 3.0.0. (ToxRat Solutions® GmbH, Germany).

The fact that the TOC concentration and the concentration (based on sebacate) measured in the solution incubated without algae (3.2 mg/L) were stable during the exposure indicates that sebacate was absorbed by the algal biomass . This means that the algae were exposed to the test substance during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium sebacate but to cover the worst-case-scenario, effect parameters based on TWA concentrations were also calculated.

Duration	Endpoint	Effect conc.	Nominal/Measured	Conc. based on	Basis for effect	Remarks (e.g. 95% CL)
72 h	EC10	5.5 mg/L	Meas. (TWA)	test mat.	growth rate	Lower 95% CL: 3.7 mg/L. Upper 95% CL: 7.3 mg/L
72 h	EC50	> 86 mg/L	Meas. (TWA)	test mat.	growth rate
72 h	NOEC	3.8 mg/L	Meas. (TWA)	test mat.	growth rate
72 h	EC10	1.1 mg/L	Meas. (TWA)	test mat.	other: Yield	Lower 95% CL: 0.5 mg/L. Upper 95% CL: 1.9 mg/L
72 h	EC50	13 mg/L	Meas. (TWA)	test mat.	other: Yield	Lower 95% CL: 9.4 mg/L. Upper 95% CL: 19 mg/L
72 h	NOEC	0.47 mg/L	Meas. (TWA)	test mat.	other: Yield

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by an average factor of >16 in 3 days, the mean coefficient of variation was 22% and the coefficient of variation of average specific growth rates in the control over the whole test was 3.9%.

Conclusions:

The 72 hr EC50 for growth inhibition was greater than the nominal concentration of 100 mg/L. The 72 hr EC50 for yield inhibition was 22 mg/L with a 95% confidence interval ranging from 17 to 28 mg/L (nominal concentrations). The 72h NOEC for growth rate inhibition was 10 mg/L based on nominal concentrations. The 72h-NOEC for yield inhibition was 1.0 mg/L based on nominal concentrations.

Executive summary:

The effect of dilithium sebacate on the growth of the algae (Pseudokirchneriella subcapitata) was investigated according to an OECD 201 guideline and EC method C3. A 72 hour test was conducted at nominal concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L dilithium sebacate. All test solutions were clear and colourless. The lithium concentration was between 97-104% of nominal throughout the test. During the first 24 hours of exposure, in the test solutions containing algae, the fatty acid (sebacate) was between 83 and 114% nominal but at the end of the test it was between 0 and 89% nominal with the loss of sebacate being proportionally higher at low concentrations. In the test solution (3.2 mg/L) without algae the sebacate concentration was 100% nominal compared with being below the limit of quantification in the test solution with algae. The total organic carbon (TOC) measured in blank solutions without algae were stable (TOC was not measured between 0.1 and 1.0 mg/L dilithium sebacate because it was below the limit of detection for the method). The fact that the TOC concentrations and the concentration (based on sebacate) measured in the solution incubated without algae (3.2 mg/L) were stable during the exposure indicates that sebacate was adsorbed by the algal biomass. This means that the algae were exposed to the test substance during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium sebacate. To cover the worst-case-scenario, effect parameters based on time weighted average (TWA) concentrations of sebacate were also calculated. The TWA concentrations were 0.050, 0.19, 0.47, 1.3, 3.8, 27 and 86 mg/L for the nominal, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L dilithium sebacate, test solutions.

The study met all the validity criteria for the test. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control. Statistically significant inhibition of growth was observed at nominal concentrations of 10 mg/L dilithium sebacate based on growth rate. The effects at 10 mg/L dilithium sebacate were <10% different from the growth of the control alage and were therefore considered to be biologically not significant. There were significant effects on yield at 3.2 mg/L dilithium sebacate. The 72 h NOEC based on growth rate was therefore 10 mg/L dilithium sebacate and the NOEC based on yield was 1.0 mg/L dilithium sebacate. Using nominal concentrations of dilithium sebacate the EC50 based on growth rate (72h-ErC50) was >100 mg/L and on yield (72h-EyC50) was 22 mg/L (CI 17-28 mg/L).

The results based on TWA concentrations of sebacate were a NOEC of 3.8 mg/L for growth rate and a NOEC of 0.47 mg/L for yield. The EC50 based on growth rate (72h-ErC50) was >86 mg/L and on yield (72h-EyC50) was 13 mg/L (CI 9.4-19 mg/L).

.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is GLP compliant and follows OECD guideline 201. Therefore, it has been given a reliability score of 1.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

See principles of method if other than guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

See principles of method if other than guideline

Principles of method if other than guideline:

Full chemical analysis was not conducted in the range finding test but as the results are based on a final definitive test with full supporting analysis this was not considered to affect the validity of the test. In the final test, cells from stock culture were used for inoculation of test solutions because the pre-culture showed insufficient growth. The validity criteria were met in the final study and study integrity was not adversely affected.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

The confimatory chemical analysis was based on the concentration of the fatty acid component (sebacate) and on the concentration of the lithium ion.Samples for analysis were taken from all test concentrations at the start, after 24 and 72 hours of exposure (lithium and sebacate analysis) and at the test start and after 72 hours of exposure (TOC analysis). Samples were stored in a refrigerator (TOC) or freezer (lithium and sebacate) 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 an intermediate 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.
For the determination of the test substance based on sebacate, the samples were diluted in a 1:1 (v:v) ratio with 2% formic acid in acetonitrile and analysed. If necessary, the samples were further diluted with 1% formic acid in 50/50 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range.
For the determination of the test substance based on lithium the samples were diluted in a 24:1 (v:v) ratio with HNO3 and analysed. If necessary, the samples were further diluted with 4% HNO3 in M2-medium to obtain concentrations within the calibration range.

Vehicle:

no

Details on test solutions:

Nominal concentrations 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg dilithium sebacate/L.
Medium: M2
Solubility: The test item was completely soluble in test medium at the concentrations tested.
Preparation of test solutions: A solution with a concentration of 100 mg/L was prepared by 20-50 minutes magnetic stirring and 6-8 minutes ultrasonic treatment. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
Appearance: The final test solutions were all clear and colourless.

Test organisms (species):

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

Details on test organisms:

Strain: NIVA CHL 1 from an in-house laboratory culture
Final test inoculation: Cells taken from stock culture
Stock culture: M1 growth medium inoculated with algal cells from pure culture on agar. Suspensions were continuously aerated and exposed to light at 21-24°C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

24 mg CaCO3/L

Test temperature:

22-24°C

pH:

7.9-8.2

Dissolved oxygen:

No data reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg Dilithium sebacate/L. For measured concentrations, please see additional information box.
The fact that the TOC concentrations and the concentration measured in the solution incubated without algae (3.2 mg/L) were stable during the exposure indicates that sebacate was absorbed by the algal biomass rather than degraded. This means that the algae were exposed to sebacate during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium sebacate. To cover the worst-case-scenario, effect parameters based on TWA concentrations were also calculated.

Details on test conditions:

Controls: Blank (test medium without test substance)
Solutions: 50 mL test solutions with 1 mL algae suspension (cell density of 10 (4) cells/mL)
Replicates: 3 per test concentration, 6 for control, 1 per test concentration for 24 hour sampling, 4 per group without algae for TOC analysis
Test vessels: 100 mL, all glass, capped
Illumination: Continuous
Incubation: Vessels were randomly distributed in the incubator and daily repositioned.
Agitation: Continuous shaking.
Observations: At the beginning of the test, cells were counted using a microscope and counting chamber and thereafter, cell densities were determined by spectrographic measurement, with algal medium as the blank. Microscopic observations were performed at the two highest test concentrations at the end of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Lower 95% CL: 7.5 mg/L. Upper 95% CL: 13 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Lower 95% CL: 1.6 mg/L. Upper 95% CL: 5.2 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Lower 95% CL: 17 mg/L. Upper 95% CL: 28 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Growth rates were in the range of the controls at the concentrations from 0.10 to 3.2 mg/L during the 72-hour test period. Statistically significant inhibition of growth rate was found at test concentrations of 10 mg/L and higher. Although a significant effect was determines at 10 mg/L dilithiumsebacate the observed effect was less than 10% of the control and was considered to be biologically insignificant. The NOEC for growth rate inhibition was therefore considered to be 10 mg/L dilithium sebacate.
Inhibition of growth rate decreased as exposure progressed.
Statistically significant inhibition of yield was found at test concentrations of 3.2 mg/L and higher so the NOEC for yield inhibition was set at 1.0 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher. The growth rate 72h-EC50 was 1.3 mg/L (95% confidence interval: 1.2 to 1.5 mg/L). The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L, so result corresponds with this range. The yield inhibition 72h-EC50 was 0.46 mg/L (95% confidence interval: 0.43 to 0.49 mg/L). The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L so result was within the low end of this range.

Reported statistics and error estimates:

Quantification of cell densities was based on a calibration curve. The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass, which is compared with the control value and the percentage inhibition in growth rate is calculated.
In case concentrations measured were below the limit of detection or the limit of quantification, the final exposure concentration(s) were taken as a factor of 2 below the applicable limit.
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 inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test substance.
The calculations were performed with ToxRat Professional v. 3.0.0. (ToxRat Solutions® GmbH, Germany).

The fact that the TOC concentration and the concentration (based on sebacate) measured in the solution incubated without algae (3.2 mg/L) were stable during the exposure indicates that sebacate was absorbed by the algal biomass . This means that the algae were exposed to the test substance during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium sebacate but to cover the worst-case-scenario, effect parameters based on TWA concentrations were also calculated.

Duration	Endpoint	Effect conc.	Nominal/Measured	Conc. based on	Basis for effect	Remarks (e.g. 95% CL)
72 h	EC10	5.5 mg/L	Meas. (TWA)	test mat.	growth rate	Lower 95% CL: 3.7 mg/L. Upper 95% CL: 7.3 mg/L
72 h	EC50	> 86 mg/L	Meas. (TWA)	test mat.	growth rate
72 h	NOEC	3.8 mg/L	Meas. (TWA)	test mat.	growth rate
72 h	EC10	1.1 mg/L	Meas. (TWA)	test mat.	other: Yield	Lower 95% CL: 0.5 mg/L. Upper 95% CL: 1.9 mg/L
72 h	EC50	13 mg/L	Meas. (TWA)	test mat.	other: Yield	Lower 95% CL: 9.4 mg/L. Upper 95% CL: 19 mg/L
72 h	NOEC	0.47 mg/L	Meas. (TWA)	test mat.	other: Yield

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by an average factor of >16 in 3 days, the mean coefficient of variation was 22% and the coefficient of variation of average specific growth rates in the control over the whole test was 3.9%.

Conclusions:

The 72 hr EC50 for growth inhibition was greater than the nominal concentration of 100 mg/L. The 72 hr EC50 for yield inhibition was 22 mg/L with a 95% confidence interval ranging from 17 to 28 mg/L (nominal concentrations). The 72h NOEC for growth rate inhibition was 10 mg/L based on nominal concentrations. The 72h-NOEC for yield inhibition was 1.0 mg/L based on nominal concentrations.

Executive summary:

The effect of dilithium sebacate on the growth of the algae (Pseudokirchneriella subcapitata) was investigated according to an OECD 201 guideline and EC method C3. A 72 hour test was conducted at nominal concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L dilithium sebacate. All test solutions were clear and colourless. The lithium concentration was between 97-104% of nominal throughout the test. During the first 24 hours of exposure, in the test solutions containing algae, the fatty acid (sebacate) was between 83 and 114% nominal but at the end of the test it was between 0 and 89% nominal with the loss of sebacate being proportionally higher at low concentrations. In the test solution (3.2 mg/L) without algae the sebacate concentration was 100% nominal compared with being below the limit of quantification in the test solution with algae. The total organic carbon (TOC) measured in blank solutions without algae were stable (TOC was not measured between 0.1 and 1.0 mg/L dilithium sebacate because it was below the limit of detection for the method). The fact that the TOC concentrations and the concentration (based on sebacate) measured in the solution incubated without algae (3.2 mg/L) were stable during the exposure indicates that sebacate was adsorbed by the algal biomass. This means that the algae were exposed to the test substance during the entire exposure period and it is justified to express the effect parameters based on nominal concentrations of dilithium sebacate. To cover the worst-case-scenario, effect parameters based on time weighted average (TWA) concentrations of sebacate were also calculated. The TWA concentrations were 0.050, 0.19, 0.47, 1.3, 3.8, 27 and 86 mg/L for the nominal, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L dilithium sebacate, test solutions.

The study met all the validity criteria for the test. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control. Statistically significant inhibition of growth was observed at nominal concentrations of 10 mg/L dilithium sebacate based on growth rate. The effects at 10 mg/L dilithium sebacate were <10% different from the growth of the control alage and were therefore considered to be biologically not significant. There were significant effects on yield at 3.2 mg/L dilithium sebacate. The 72 h NOEC based on growth rate was therefore 10 mg/L dilithium sebacate and the NOEC based on yield was 1.0 mg/L dilithium sebacate. Using nominal concentrations of dilithium sebacate the EC50 based on growth rate (72h-ErC50) was >100 mg/L and on yield (72h-EyC50) was 22 mg/L (CI 17-28 mg/L).

The results based on TWA concentrations of sebacate were a NOEC of 3.8 mg/L for growth rate and a NOEC of 0.47 mg/L for yield. The EC50 based on growth rate (72h-ErC50) was >86 mg/L and on yield (72h-EyC50) was 13 mg/L (CI 9.4-19 mg/L).

.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 11 March 2010 and 26 March 2010.

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.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date for Inspection: 15/09/2009 Date for signature: 26/11/2009

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
100 mg/l loading rate WAF test group

- Sampling method:
Samples
were taken from the uninoculated control and 100 mg/l loading rate WAF test group at 0 and 72 hours for this analysis

- Sample storage conditions before analysis:
Duplicate samples were taken and stored frozen (approximately -20 degC) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
An amount of test item (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/I loading rate. After the addition of the test item, 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 mixture 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 em 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 middepth siphoning (the first 75-100 ml discarded) to give the 100 mg/I loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (12.5 ml) to give the required test concentration of 100 rng/l loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the uninoculated test preparations at 0 and 72 hours.

- Eluate:
Not applicable
- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item 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.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
No

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Algae

- Strain:
Strain CCAP 276/20

- Source:
Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

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 10E3 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 deg C until the algal cell density was approximately 10E4 - 10E5 cells/ml.

ACCLIMATION

- Acclimation period:
Not recorded.

- Culturing media and conditions:

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Any deformed or abnormal cells observed:
None recorded.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not recorded.

Test temperature:

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

pH:

7.3 - 7.8
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:

Range-finding Test: 10 and 100 mg/l (nominal)

Definitive Test: 100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 100ml
- 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: 10E3 cells/ml

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

- No. of organisms per vessel:
initial cell density of approximately 10E4- 10E5 cells/ml.

- No. of vessels per concentration (replicates):
two 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.

TEST MEDIUM
An amount of test item (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/I loading rate. After the addition of the test item, 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 mixture 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 em 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 middepth siphoning (the first 75-100 ml discarded) to give the 100 mg/I loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (12.5 ml) to give the required test concentration of 100 rng/l loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the uninoculated test preparations at 0 and 72 hours.



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 completely filled with test preparation were used for the control and three flasks each completely filled 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 3.21 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.
The flasks were sealed with ground glass stoppers 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, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Determination of ECx values
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). ECxvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949). 


- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS

- Range finding study:
10 and 100 mg/l loading rate WAF.

-Definitive Test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 100 mg/l.

The control group was maintained under identical conditions but not exposed to the test material.

VALIDATION:
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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: 95% CL not stated

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and yield

Remarks on result:

other: 95% CL not stated

Details on results:

- Exponential growth in the control (for algal test): yes

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 and following a 1-Hour settlement period the WAF was observed to have formed a clear colourless media column with large lumps of test item floating at the media surface.

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 test cultures were observed to be pale green dispersions.

- Any stimulation of growth found in any treatment:
None

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
None recorded

Growth rate Data
The growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/I.

Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL*10(0 - 72 h)           : >100 mg/l loading rate WAF
ErL*20(0 - 72 h)           : >100 mg/l loading rate WAF
ErL*50(0 - 72 h)           : >100 mg/l loading rate WAF

where ErL*xis the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/I loading rate WAF test group using a Student's t-test 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 (P ≥0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/iloading rate WAF.

* EL =Effective Loading Rate

Inhibition of yield
EyL*10(0 - 72 h)           : >100 mg/l loading rate WAF
EyL*20(0 - 72 h)           : >100 mg/l loading rate WAF
EyL*50(0 - 72 h)           : >100 mg/l loading rate WAF

where EyL*xis the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as indicated above. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P ≥0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/iloading rate WAF.

Results with reference substance (positive control):

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:

ErC50 (0 - 72 h) : 0.49* mg/l
EyC50 (0 - 72 h) : 0.18 mg/I, 95% confidence limits 0.16 - 0.21 mg/I

No Observed Effect Concentration (NOEC) based on growth rate : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l

Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 mg/l

The results from the positive control with potassium dichromate were within the normal range for this reference material.

* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Reported statistics and error estimates:

A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/I loading rate 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).

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Desmodesmus subspicatus has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

* EL = Effective Loading Rate

Executive summary:

Introduction. A study was performed to assess the effect of the test item 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. Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) 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 item gave EL*50 values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of test item in the WAF.

Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

* EL =Effective Loading Rate

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Between 11 March 2010 and 26 March 2010.

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.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date for Inspection: 15/09/2009 Date for signature: 26/11/2009

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
100 mg/l loading rate WAF test group

- Sampling method:
Samples
were taken from the uninoculated control and 100 mg/l loading rate WAF test group at 0 and 72 hours for this analysis

- Sample storage conditions before analysis:
Duplicate samples were taken and stored frozen (approximately -20 degC) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
An amount of test item (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/I loading rate. After the addition of the test item, 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 mixture 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 em 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 middepth siphoning (the first 75-100 ml discarded) to give the 100 mg/I loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (12.5 ml) to give the required test concentration of 100 rng/l loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the uninoculated test preparations at 0 and 72 hours.

- Eluate:
Not applicable
- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item 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.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
No

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Algae

- Strain:
Strain CCAP 276/20

- Source:
Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

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 10E3 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 deg C until the algal cell density was approximately 10E4 - 10E5 cells/ml.

ACCLIMATION

- Acclimation period:
Not recorded.

- Culturing media and conditions:

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Any deformed or abnormal cells observed:
None recorded.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not recorded.

Test temperature:

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

pH:

7.3 - 7.8
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:

Range-finding Test: 10 and 100 mg/l (nominal)

Definitive Test: 100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 100ml
- 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: 10E3 cells/ml

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

- No. of organisms per vessel:
initial cell density of approximately 10E4- 10E5 cells/ml.

- No. of vessels per concentration (replicates):
two 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.

TEST MEDIUM
An amount of test item (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/I loading rate. After the addition of the test item, 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 mixture 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 em 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 middepth siphoning (the first 75-100 ml discarded) to give the 100 mg/I loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (12.5 ml) to give the required test concentration of 100 rng/l loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the uninoculated test preparations at 0 and 72 hours.



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 completely filled with test preparation were used for the control and three flasks each completely filled 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 3.21 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.
The flasks were sealed with ground glass stoppers 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, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Determination of ECx values
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). ECxvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949). 


- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS

- Range finding study:
10 and 100 mg/l loading rate WAF.

-Definitive Test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 100 mg/l.

The control group was maintained under identical conditions but not exposed to the test material.

VALIDATION:
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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: 95% CL not stated

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and yield

Remarks on result:

other: 95% CL not stated

Details on results:

- Exponential growth in the control (for algal test): yes

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 and following a 1-Hour settlement period the WAF was observed to have formed a clear colourless media column with large lumps of test item floating at the media surface.

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 test cultures were observed to be pale green dispersions.

- Any stimulation of growth found in any treatment:
None

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
None recorded

Growth rate Data
The growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/I.

Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL*10(0 - 72 h)           : >100 mg/l loading rate WAF
ErL*20(0 - 72 h)           : >100 mg/l loading rate WAF
ErL*50(0 - 72 h)           : >100 mg/l loading rate WAF

where ErL*xis the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/I loading rate WAF test group using a Student's t-test 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 (P ≥0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/iloading rate WAF.

* EL =Effective Loading Rate

Inhibition of yield
EyL*10(0 - 72 h)           : >100 mg/l loading rate WAF
EyL*20(0 - 72 h)           : >100 mg/l loading rate WAF
EyL*50(0 - 72 h)           : >100 mg/l loading rate WAF

where EyL*xis the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as indicated above. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P ≥0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/iloading rate WAF.

Results with reference substance (positive control):

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:

ErC50 (0 - 72 h) : 0.49* mg/l
EyC50 (0 - 72 h) : 0.18 mg/I, 95% confidence limits 0.16 - 0.21 mg/I

No Observed Effect Concentration (NOEC) based on growth rate : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l

Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 mg/l

The results from the positive control with potassium dichromate were within the normal range for this reference material.

* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Reported statistics and error estimates:

A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/I loading rate 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).

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Desmodesmus subspicatus has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

* EL = Effective Loading Rate

Executive summary:

Introduction. A study was performed to assess the effect of the test item 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. Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) 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 item gave EL*50 values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of test item in the WAF.

Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

* EL =Effective Loading Rate

Description of key information

This endpoint is completed using data read across from structurally similar substances, dilithium sebacate and fatty acids, lanolin.

For dilithium sebacate, the 72 hr EC50 for growth inhibition was > 100 mg/L and for yield inhibition was 22 mg/L. The 72h NOEC for growth rate inhibition was 10 mg/L and for yield inhibition was 1.0 mg/L. For fatty acids, lanolin, the 72 h EL50 values for growth rate and yield were > 100 mg/L loading rate WAF and correspondingly the NOELR was 100 mg/L loading rate WAF.

Additional information

No data are available for the substance fatty acids, lanolin, lithium salts. Read across from dilithium sebacate and fatty acids, lanolin is used to complete the toxicity to aquatic algae endpoint.

Two key studies conducted with read across substances, dilithium sebacate and fatty acids, lanolin are available for this endpoint.

The effect of dilithium sebacate on the growth of the algae (Pseudokirchneriella subcapitata) was investigated in a study following OECD guideline 201. The test was conducted at nominal concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L dilithium sebacate for 72 hours. The 72 hr EC50 for growth inhibition was > 100 mg/L and for yield inhibition was 22 mg/L. The 72h NOEC for growth rate inhibition was 10 mg/L and for yield inhibition was 1.0 mg/L.

The effect of fatty acids, lanolin on the growth of the green alga Desmodesmus subspicatus was investigated in a study conducted according to OECD guideline 201. Following a preliminary range-finding test, green algae was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L for 72 hours. The 72 h EL50 values for growth rate and yield were > 100 mg/L loading rate WAF and correspondingly the NOELR was 100 mg/L loading rate WAF.

The studies were conducted according to the OECD 201 guideline and EC method C.3 and are GLP-compliant, therefore are considered reliable and suitable for this endpoint.