1,2,3,4,4a,7,8,8a-octahydro-2,4a,5,8a-tetramethyl-1-naphthyl formate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 07 April 2014 and 14 May 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: SC00010054
Purity: >96.5%
Appearance / Physical Description: Clear colorless liquid

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary. Samples and duplicates of each uninoculated test preparation were taken at 72 hours and stored frozen for analysis.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature within the incubator was recorded daily.

pH:

The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter.

Nominal and measured concentrations:

Range-finding test - nominal concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution
Definitive test - nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution. Analysis of the test preparations at 0 hours showed measured test concentrations of the 32, 56 and 100 % v/v saturated solutions as 1.25, 2.11 and 3.33 mg/L respectively. The corresponding geometric mean measured concentrations (0-72h) were 1.08, 1.83 and 3.16 mg/L.

Details on test conditions:

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.

Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that it was not possible to prepare a 100 mg/L stock solution in water using traditional methods of preparation e.g. ultrasonication.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions .

Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 3 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
An amount of test item (nominally 2250 mg) was dispersed in 22.5 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 0.10, 1.0 and 10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
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 used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item.
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 ± 1 ºC 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.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100% v/v saturated solution.

Experimental Preparation
An amount of test item (2250 mg) was dispersed in 22 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
A series of dilutions was made from this saturated solution to give stock solutions of 10, 18, 32 and 56% v/v saturated solution. An aliquot (600 mL) of each of the stock solutions was inoculated with 5.0 mL of algal suspension to give the required test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution 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 item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.05 x 105 cells per mL. Inoculation of 600 mL of test medium with 5.0 mL of this algal suspension gave an initial nominal cell density of 5 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.

Evaluations
Test Organism Observations
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 9% inhibition at highest test concentration

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 3.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 9% inhibition at highest test concentration

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Effects based on yield were also reported (see attached report and below summary). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on test design (ECHA guidance chapter R.7b v2.0, OECD 201 Guideline). The guideline includes the additional response yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaptation 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table.

Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.
Based on this information test concentrations of 10, 18, 32, 56, and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours (see Appendix 5) showed measured concentration of 0.475 and 3.30 mg/L and at 72 hours showed measured concentration of less than the Limit of Quantification and 0.0772 mg/L indicating that the test item was either unstable and/or was adsorbing to the algal cells present.

Definitive Test
Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.25 to 3.33 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.0274 and 0.612 mg/L (1.3% to 18% of the 0-Hour measured test concentrations). Analysis of uninoculated aged test preparations at 72 hours gave measured concentrations of 0.934 to 3.00 mg/L, indicating that the test item adsorbed to the algal biomass in the test suspensions.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations of the 0-Hour and 72-Hour uninoculated aged test preparations

Growth Data
From the data, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item at a geometric mean measured test concentration of 3.16 mg/L over the 72-Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h): >3.16 mg/L
ErC20 (0 - 72 h): >3.16 mg/L
ErC50 (0 - 72 h): >3.16 mg/L*
* It was not possible to calculate 95% confidence limits for the EC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Where:
ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations 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). The highest test concentration where there was no statistically significant difference with the control was the 1.83 mg/L test concentration (P≥0.05), therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 1.83 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 3.16 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 2.6 mg/L
EyC20 (0 - 72 h): 2.9 mg/L
EyC50 (0 - 72 h): >3.16 mg/L*
* It was not possible to calculate 95% confidence limits for the EC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Where:
EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 4.2.2.1. The highest test concentration where there was no statistically significant difference with the control was the 1.83 mg/L test concentration (P≥0.05), therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 1.83 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 3.16 mg/L.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 10, 18, 32 and 56% v/v saturated solution, however cell debris was observed to be present in the test cultures at 100% v/v saturated solution.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41303826) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.1 mg/L; 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.51 mg/L; 95% confidence limits 0.45 – 0.59 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 item.

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 concentrations 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).

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a % of the 0-Hour Measured Test Concentration
32	1.08	86
56	1.83	87
100	3.16	95

 

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 85 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 : 4.45 x 103cells per mL

Mean cell density of control at 72 hours : 3.81 x 105cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% 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 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Water Quality Criteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 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.

 

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 10, 18, 32 and 56% v/v saturated solution test preparations were green dispersions whereas the 100% v/v saturated solution test preparations were pale green dispersions.

Green coloration of the test preparations occurs over the test period due to the growth of the algal cells present, the depth of coloration depending upon the increase in cell density.

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

 

Nominal Concentration (% v/v Saturated Solution)		Cell Densities* (cells per mL)		Inhibition Values (%)
		Hours	72 Hours	Growth Rate	Yield
Control	R1	5.25E+03	1.02E+06	-	-
	R2	5.50E+03	8.96E+05
	Mean	5.38E+03	9.44E+05
0.1	R1	6.37E+03	9.11E+05	4	3
	R2	6.54E+03	9.18E+05
	Mean	6.46E+03	9.15E+05
1.0	R1	5.07E+03	1.07E+06	[3]	[14]
	R2	5.77E+03	1.08E+06
	Mean	5.37E+03	1.07E+06
10	R1	4.99E+03	1.06E+06	[1]	[14]
	R2	6.22E+03	1.10E+06
	Mean	5.61E+03	1.08E+06
100	R1	5.63E+03	7.79E+05	3	13
	R2	4.86E+03	8.62E+05
	Mean	5.25E+03	8.21E+05

*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

[Increase in growth compared to controls]

Cell Densities and pH Values in the Definitive Test

Nominal Concentration (% v/v Saturate Solution)		pH	Cell Densities* (cells per mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.7	5.81E+03	2.29 E+04	9.8 E+04	4.00 E+05	7.9
	R2		5.94 E+03	2.52 E+04	1.08 E+05	4.33 E+05
	R3		3.94 E+03	2.16 E+04	1.02 E+05	3.46 E+05
	R4		3.57 E+03	2.72 E+04	8.30 E+04	3.95 E+05
	R5		3.62 E+03	2.20 E+04	7.62 E+04	3.92 E+05
	R6		3.86 E+03	2.11 E+04	6.91 E+04	3.18 E+05
	Mean		4.45 E+03	2.33 E+04	8.96 E+04	3.81 E+05
10	R1	7.8	4.08 E+03	2.52 E+04	8.91 E+04	5.32 E+05	8.1
	R2		3.90 E+03	3.25 E+04	1.08 E+04	5.04 E+05
	R3		3.89 E+03	2.82 E+04	1.05 E+05	4.35 E+05
	Mean		3.96 E+03	2.86 E+04	1.01 E+05	4.90 E+05
18	R1	7.8	4.06 E+03	2.12 E+04	9.13 E+04	4.51 E+05	8.1
	R2		3.92 E+03	2.30 E+04	9.08 E+04	3.90 E+05
	R3		4.16 E+03	2.19 E+04	8.36 E+04	3.48 E+05
	Mean		4.05 E+03	2.20 E+04	8.86 E+04	3.96 E+05
32	R1	7.8	4.48 E+03	2.21 E+04	7.57 E+04	3.67 E+05	8.0
	R2		4.26 E+03	2.14 E+04	8.93 E+04	4.85 E+05
	R3		4.29 E+03	1.89 E+04	8.42 E+04	4.66 E+05
	Mean		4.34 E+03	2.08 E+04	8.31 E+04	4.39 E+05
56	R1	7.8	4.16 E+03	1.93 E+04	7.41 E+04	3.83 E+05	8.0
	R2		4.21 E+03	1.68 E+04	7.39 E+04	3.89 E+05
	R3		4.10 E+03	2.10 E+04	7.50 E+04	3.61 E+05
	Mean		4.15 E+03	1.90 E+04	7.43 E+04	3.78 E+05
100	R1	7.8	4.00 E+03	1.27 E+04	5.14 E+04	2.59 E+05	7.8
	R2		3.66 E+03	1.28 E+04	4.71 E+04	2.53 E+05
	R3		3.87 E+03	1.32 E+04	4.34 E+04	2.50 E+05
	Mean		3.85 E+03	1.29 E+04	4.73 E+04	2.54 E+05

*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

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	R2	0.067	0.061	0.058
	R3	0.061	0.065	0.051
	R4	0.071	0.046	0.065
	R5	0.062	0.052	0.068
	R6	0.060	0.049	0.064
	Mean	0.064	0.056	0.061

  R1 - R6 = Replicates 1 to 6

Inhibition of Growth Rate and Yield in the Definitive Test

 

Nominal Concentration (% v/v Saturated Solution)		Growth rate (cells/mL/hour)		Yield (cells / mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition
Control	R1	0.061	-	3.95E+05	-
	R2	0.062		4.27 E+05
	R3	0.059		3.42 E+05
	R4	0.061		3.91 E+05
	R5	0.061		3.89 E+05
	R6	0.058		3.14 E+05
	Mean	0.060		3.76 E+05
	SD	0.002		4.07 E+04
10	R1	0.065	[8]	5.28 E+05
	R2	0.064	[7]	5.00 E+05
	R3	0.062	[3]	4.31 E+05
	Mean	0.3064	[6]	4.86 E+05	[29]
	SD	0.002		5.00 E+05
18	R1	0.063	[5]	4.47 E+05
	R2	0.060	0	3.86 E+05
	R3	0.059	2	3.44 E+05
	Mean	0.061	[1]	3.92 E+05	[4]
	SD	0.002		5.18 E+04
32	R1	0.060	0	3.62 E+05
	R2	0.064	[7]	4.80 E+05
	R3	0.063	[5]	4.62 E+05
	Mean	0.062	[4]	4.35 E+05	[16]
	SD	0.002		6.36 E+04
56	R1	0.060	0	3.79 E+05
	R2	0.060	0	3.85 E+05
	R3	0.059	2	3.57 E+05
	Mean	0.060	1	3.74 E+05	1
	SD	0.001		1.46 E+04
100	R1	0.055	8	2.55 E+05
	R2	0.054	10	2.49 E+05
	R3	0.054	10	2.46 E+05
	Mean	0.054	9	2.50 E+05	34
	SD	0.001		4.22 E+03

* 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

[Increase in growth as compared to controls]

Analytical investigations

Preparation of Test Samples

Nominal Concentration of Test Item [% v/v Saturated Solution]	Sample Volume [mL]	Final Volume [mL]	Sample Preparation Factor F
Control	200	20	0.1
32	200	20	0.1
56	200	20	0.1
100	200	20	0.1

 

 

Linearity Data

Concentration of Test Item [mg/L]	Mean Area [counts]
0	0
0.526	1.753 x 105
1.05	3.211 x 105
5.26	1.573 x 106
10.5	3.380 x 106
10.6	3.333 x 106
21.0	6.706 x 106
42.1	1.397 x 107
63.1	2.018 x 107

 

 

Results for Range-Finding Samples

Timepoint [hours]	Nominal concentration of test item Cnom [% v/v saturated solution]	Measured concentration of test item in sample vial X [mg/L]	Sample preparation factor F	Determined concentration of test item C [mg/L]
0	10	4.75	0.1	0.475
	100	33.0	0.1	3.30
72	10	<LOQ	0.1	<LOQ
	100	0.772	0.1	0.0772

 

Results for spiked recovery samples

Nominal concentration of test item Cnom   [mg/L]	Fortified concentration of test item in the spiked sample Cfort [mg/L]	Measured concentration of test item in the sample vial X [mg/L]	Sample preparation facto F	Determined concentration of test item in the spiked sample C [mg/L]	Mean analytical recovery rate R [%]	Precision (relative standard deviation of recovery) [%]
0.10	0.102	1.04	0.1	0.104	101	1.3
	0.102	1.01	0.1	0.101
	0.102	1.02	0.1	0.102
	0.102	1.04	0.1	0.104
	0.102	1.03	0.1	0.103
3.0	3.06	27.6	0.1	2.76	92	1.4
	3.06	28.0	0.1	2.80
	3.06	28.2	0.1	2.82
	3.06	28.5	0.1	2.85
	3.06	28.6	0.1	2.86
Acceptance target					80 – 120	<10

For reporting purposes, x has been calculated from c retrospectively.

LOQ = Limit of Quantification

 

Results for test samples

Timepoint [hours]	Nominal concentration of test item Cnom [% v/v saturated solution]	Measured concentration of test item in sample vial X [mg/L]	Sample preparation factor F	Determined concentration of test C [mg/L]
0 (fresh)	Control	< LOQ	0.1	< LOQ
	32	12.5	0.1	1.25
	56	21.1	0.1	2.11
	100	33.3	0.1	3.33
72 (old)	Control	< LOQ	0.1	< LOQ
	32	1.34	0.1	0.134
	56	0.274	0.1	0.0274
	100	6.12	0.1	0.612
72 (old) uninoculated	32	9.34	0.1	0.934
	56	16.0	0.1	1.60
	100	30.0	0.1	3.00

LOQ = Limit of quantification

For reporting purposes, x has been calculated from c retrospectively.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC10, EC20 and EC50 values for growth rate of greater than 3.16 mg/L based on the geometric mean measured test concentrations (9% inhibition seen at highest test concentration). For yield, the EC10, EC20 and EC50 values were determined as 2.6, 2.9 and greater than 3.16 mg/L respectively based on the geometric mean measured test concentrations (34% inhibition at highest test concetration). The No Observed Effect Concentration for growth rate and yield was determined as 1.83 mg/L. Correspondingly the Lowest Observed Effect Concentration for growth rate and yield was 3.16 mg/L.
The preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on test design (ECHA guidance chapter R.7b v2.0, OECD 201 Guideline). The guideline includes the additional response yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaptation 286/2011) also states that classification should be based on the ErC50. Thus the 72-h EC50 based on growth rate and corresponding are used for classification purposes, which were determined in this study to be >3.16 mg/L and 1.83 mg/L respectively.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. 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) No 761/2009.

 

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 3 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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

Analysis of the 32, 56 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 1.25 to 3.33 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.0274 and 0.612 mg/L (1.3% to 18% of the 0-Hour measured test concentrations). Analysis of uninoculated aged test preparations at 72 hours gave measured concentrations of 0.934 to 3.00 mg/L, indicating that the test item adsorbed to the algal biomass in the test suspensions. It was therefore considered appropriate to calculate the results based on the geometric mean measured test concentration of the 0-Hour and 72-Hour uninoculated aged test preparations only.

Exposure ofPseudokirchneriella subcapitatato the test item gave EC10, EC20and EC50values for growth rate of greater than 3.16 mg/L based on the geometric mean measured test concentrations. For yield, the EC10, EC20and EC50values were determined as 2.6, 2.9 and greater than 3.16 mg/L respectively based on the geometric mean measured test concentrations.

The No Observed Effect Concentration for growth rate and yield was determined as 1.83 mg/L. Correspondingly the Lowest Observed Effect Concentration for growth rate and yield was 3.16 mg/L.

Description of key information

The short term toxicity to aquatic algae of the test substance was assessed according to OECD Guideline for Testing of Chemicals No. 201 using an algal growth inhibition method.  The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50.Thus the 72-h EC50 and NOEC based on growth rate, which were determined in this study to be >3.16 mg/L and 1.83 mg/L respectively, are used for classification purposes and have been selected as the key values for chemical safety assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.16 mg/L

EC10 or NOEC for freshwater algae:

1.83 mg/L

Additional information

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication. A dissolved test item concentration of approximately 3 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. The solutions were prepared by stirring an excess of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.  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.

Analysis of the 32, 56 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 1.25 to 3.33 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.0274 and 0.612 mg/L (1.3% to 18% of the 0-Hour measured test concentrations). Analysis of uninoculated aged test preparations at 72 hours gave measured concentrations of 0.934 to 3.00 mg/L, indicating that the test item adsorbed to the algal biomass in the test suspensions.Given that disappearance of the test substance from solution by adsorption to the algal biomass does not mean that it is lost from the test system and that the algae are still exposed to the test substance, it was considered appropriate to calculate the results based on the geometric mean measured test concentration of the 0-Hour and 72-Hour uninoculated aged test preparations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC10, EC20 and EC50 values for growth rate of greater than 3.16 mg/L based on the geometric mean measured test concentrations. For yield, the EC10, EC20 and EC50 values were determined as 2.6, 2.9 and greater than 3.16 mg/L respectively based on the geometric mean measured test concentrations. The No Observed Effect Concentration for growth rate and yield was determined as 1.83 mg/L. Correspondingly the Lowest Observed Effect Concentration for growth rate and yield was 3.16 mg/L.