Bis[(3,4-epoxycyclohexyl)methyl] adipate

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:

26 October 2017 to 12 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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)

Analytical monitoring:

yes

Details on sampling:

- Samples were taken from the control and each test group from the bulk test preparation at 0 hours, from additional samples ran alongside at 24 and 48 hours and from the pooled replicates at 72 hours for quantitative analysis.
- Duplicate samples were taken at 0, 24, 48 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
- Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document, therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
- As a result of the trial the test material was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 34 mg/L.
- Preparation of test material solutions: A nominal amount of test material (550 mg) was dispersed in 11 litres 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 material was removed by filtration through a 0.2 µm filter (first approximate 2 litres 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 56, 32, 18 and 10 % v/v saturated solution. An aliquot (1 litre) of each of the stock solutions was separately inoculated with 4.5 mL of algal suspension to give the required test concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution.
- The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
- The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Method of cultivation: The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 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 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.2 - 8.5

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100 % v/v Saturated Solutions
Geometric Mean Measured Test Concentrations: 0.32, 0.35, 3.0, 5.8 and 17 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: Closed- the flasks were plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.10 x 10^6 cells per mL. Inoculation of 1 liter of test medium with 4.5 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
- No. of vessels per concentration: 3
- No. of vessels per control: 6

TEST MEDIUM
- The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Culture medium: NaNO3 25.5 mg/L, MgCl2.6H2O 12.16 mg/L, CaCl2.2H2O 4.41 mg/L, MgSO4.7H2O 14.6 mg/L, K2HPO4 1.044 mg/L, NaHCO3 15.0 mg/L, H3BO3 0.186 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCl2 0.00327 mg/L, FeCl3.6H2O 0.160 mg/L, CoCl2.6H2O 0.00143 mg/L, Na2MoO4.2H2O 0.00726 mg/L, CuCl2.2H2O 0.000012 mg/L and Na2EDTA.2H2O 0.30 mg/L.
- The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Water Quality Criteria: 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. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
- Constantly shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED:
- Test Organism Observations: Samples were taken at 24, 45 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density. To determine the potential effect of the test material 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.
- Test material concentrations were analytically verified.

TEST CONCENTRATIONS
- Range finding study: 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.
- Results used to determine the conditions for the definitive study: yes, based on the results of the range-finding test an initial experiment was conducted at test concentrations of 18, 32, 56 and 100 % v/v saturated solution. The results obtained from the chemical analyses at 24 and 48 hours were inconsistent and as such the test was repeated.


DATA EVALUATION
- Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (ln Nn – ln N1) / (tn – t1)

Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
Percentage inhibition of growth rate for each replicate test material vessel was calculated using the following equation:

Ir = [(µc - µt) / µc] x 100

Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture

- Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn – N0

Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = [(Yc – Yt)/ Yc ] x 100

Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group

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 computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.

Geometric Mean Measured Test Concentrations
The geometric mean measured test concentrations of the samples were calculated as follows:

GM = v(C0 x C1)

Where:
GM = geometric mean measured test concentration (mg/L)
C0 = measured concentration at the start of the 24-Hour period (mg/L)
C1 = measured concentration at the end of the 24-Hour period (mg/L)

The geometric mean measured test concentrations for each 24-Hour period (0-24, 24-48 and 48-72 hours) were determined and the arithmetic mean of the three values determined. Where a measured concentration of less than the LOQ was obtained, a value of ½ the LOQ was substituted into the equation.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 17 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.:

3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: It was not possible to determine the NOEC or LOEC based on inhibition of yield as significant effects were observed at all test concentrations employed

Details on results:

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 showed measured test concentrations of 3.3 and 40 mg/L respectively were obtained. A decline in measured test concentrations was observed at 72 hours to less than the LOQ, determined to be 0.0016 mg/L indicating that the test material was unstable under the conditions of the test.


DEFINITIVE TEST
Verification of Test Concentrations
- Analysis of the test preparations at 0 hours showed measured test concentrations to range from 3.1 to 33 mg/L. A decline in measured test concentrations after each 24-Hour period was observed in the range of 0.27 to 30 mg/L at 24 hours, 0.010 to 14 mg/L at 48 hours and from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0016 mg/L, to 0.015 mg/L at 72 hours.
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 in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.00080 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations were determined to be: 0.32, 0.35, 3.0, 5.8 and 17 mg/L at nominal concentrations of 10, 18, 32, 56 and 100 % v/v Saturated Solutions, respectively.

Growth Data
- Growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test material over the 72-Hour exposure period.
- Inhibition of Growth Rate:
ErC10 (0 to 72 hour): 8.5 mg/L
ErC20 (0 to 72 hour): >17 mg/L
ErC50 (0 to 72 hour): >17 mg/L
- It was not possible to calculate ErC20 or ErC50 values as no concentration tested resulted in greater than 20 % inhibition of growth rate.
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). There were no statistically significant differences between the control, 0.32, 0.35, 3.0 and 17 mg/L test concentrations (P¿0.05), however the 5.8 mg/L test concentration was significantly different (P<0.05). A further set of analyses was run excluding the 5.8 mg/L test group data. There were no statistically significant differences between the control, 0.32, 0.35 and 3.0 mg/L test concentrations (P¿0.05), however the 17 mg/L test concentration was significantly different (P<0.05). As visual inspection of the data showed there to be greater than 10% inhibition of growth rate at both 5.8 and 17 mg/L (12 and 11% respectively) it was considered that the results of the statistical analyses were spurious and therefore the NOEC was considered to be 3.0 mg/L. Correspondingly the LOEC was considered to be 5.8 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 0.048 mg/L
EyC20 (0 to 72 hour): 0.33 mg/L
EyC50 (0 to 72 hour): >17 mg/L
- The EyC10 value was determined by extrapolation of the curve as at the lowest test concentration employed, 16 % inhibition of yield occurred. It was not possible to calculate an EyC50 value as no concentration tested resulted in greater than 44 % inhibition of yield.
- Statistical analysis of the yield data including all test groups showed there to be no statistically significant differences (P=0.05), between the control, 0.32, 0.35 and 3.0 mg/L test groups, however all other test concentrations were significantly different (P<0.05). Once more the results of the statistical analyses were considered to be spurious given that at 0.32, 0.35 and 3.0 mg/L, 16, 26 and 22 % inhibition of yield occurred respectively. It was therefore not possible to determine NOEC and LOEC values for inhibition of yield.

OBSERVATIONS
- All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

WATER QUALITY CRITERIA
- Temperature was maintained at 24 ±1 °C throughout the test.
- The pH value of the control cultures was observed to increase from pH 7.2 at 0 hours to pH 8.5 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.

TEST MATERIAL SOLUBILITY
- 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 green dispersions.

Results with reference substance (positive control):

A positive control 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 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration 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. One set of analyses were ran including all test concentrations; another set was ran excluding the 5.8 mg/L test group. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validation Criteria

- The following data show that the cell concentration of the control cultures increased by a factor of 134 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.

Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL

Mean cell density of control at 72 hours: 6.69 x 10^5 cells per mL

- The mean coefficient of variation for section by section specific growth rate for the control cultures was 16 % 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 to 72 hours) was 6 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

Table 1: 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 (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.38E+03	1.44E+06	-	-
	R2	4.91E+03	1.86E+06
	Mean	5.14E+03	1.65E+06
0.10	R1	5.04E+03	1.69E+06	1	8
	R2	5.23E+03	1.33E+06
	Mean	5.14E+03	1.51E+06
1.0	R1	4.93E+03	1.46E+06	0	7
	R2	4.88E+03	1.60E+06
	Mean	4.90E+03	1.53E+06
10	R1	5.92E+03	1.60E+06	1	2
	R1	4.74E+03	1.65E+06
	Mean	5.33E+03	1.62E+06
100	R1	4.80E+03	5.64E+05	16	66
	R2	4.67E+03	5.81E+05
	Mean	4.74E+03	5.72E+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.

R = Replicate

- = Not applicable

Table 2: Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hour	25 Hour	47 Hour	72 Hour	72 Hour
Control	R1	7.2	2.43E+04	1.52E+05	8.40E+05	8.5
	R2		1.77E+04	1.13E+05	5.83E+05
	R3		2.18E+04	1.31E+05	8.44E+05
	R4		2.53E+04	1.24E+05	4.15E+05
	R5		2.61E+04	1.04E+05	6.26E+05
	R6		2.20E+04	1.31E+05	7.08E+05
	Mean		2.29E+04	1.26E+05	6.69E+05
0.32	R1	7.3	2.28E+04	1.04E+05	4.69E+05	8.3
	R2		2.28E+04	1.13E+05	5.71E+05
	R3		2.14E+04	1.09E+05	6.47E+05
	Mean		2.23E+04	1.09E+05	5.63E+05
0.35	R1	7.3	2.24E+04	9.81E+04	5.00E+05	8.1
	R2		2.56E+04	1.08E+05	4.74E+05
	R3		1.89E+04	8.82E+04	5.23E+05
	Mean		2.23E+04	9.80E+04	4.99E+05
3.0	R1	7.3	2.63E+04	8.63E+04	4.33E+05	8.0
	R2		2.34E+04	1.22E+05	5.60E+05
	R3		2.47E+04	1.29E+05	5.71E+05
	Mean		2.48E+04	1.12E+05	5.21E+05
5.8	R1	7.3	2.12E+04	7.03E+04	3.59E+05	7.9
	R2		1.74E+04	8.32E+04	4.89E+05
	R3		1.40E+04	5.90E+04	2.89E+05
	Mean		1.75E+04	7.08E+04	3.79E+05
17	R1	7.3	1.53E+04	7.66E+04	3.65E+05	7.9
	R2		2.05E+04	8.65E+04	5.67E+05
	R3		1.11E+04	4.71E+04	2.89E+05
	Mean		1.56E+04	7.01E+04	4.07E+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

R =  Replicate

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

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.063	0.083	0.068
	R2	0.050	0.084	0.066
	R3	0.059	0.082	0.074
	R4	0.065	0.072	0.048
	R5	0.066	0.063	0.072
	R6	0.059	0.081	0.068
	Mean	0.060	0.078	0.066

R= Replicate

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

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.071	-	8.35E+05	-
	R2	0.066		5.78E+05
	R3	0.071		8.39E+05
	R4	0.061		4.10E+05
	R5	0.067		6.21E+05
	R6	0.069		7.03E+05
	Mean	0.068		6.64E+05
	SD	0.004		1.65E+05
0.32	R1	0.063	7	4.64E+05
	R2	0.066	3	5.66E+05
	R3	0.068	0	6.42E+05
	Mean	0.066	3	5.58E+05	16
	SD	0.003		8.92E+04
0.35	R1	0.064	6	4.95E+05
	R2	0.063	7	4.69E+05
	R3	0.065	4	5.18E+05
	Mean	0.064	6	4.94E+05	26
	SD	0.001		2.48E+04
3.0	R1	0.062	9	4.28E+05
	R2	0.066	3	5.55E+05
	R3	0.066	3	5.66E+05
	Mean	0.065	5	5.16E+05	22
	SD	0.002		7.68E+04
5.8	R1	0.059	13	3.54E+05
	R2	0.064	6	4.84E+05
	R3	0.056	18	2.84E+05
	Mean	0.060	12	3.74E+05	44
	SD	0.004		1.02E+05
17	R1	0.060	12	3.60E+05
	R2	0.066	3	5.62E+05
	R3	0.056	18	2.84E+05
	Mean	0.061	11	4.02E+05	39
	SD	0.005		1.44E+05

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

R = Replicate

SD = Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the geometric mean measured test concentrations: Growth rate and yield inhibition EC50 > 17 mg/L, growth rate NOEC of 3.0 mg/L and growth rate LOEC of 5.8 mg/L. It was not possible to determine the NOEC or LOEC based on inhibition of yield as significant effects were observed at all test concentrations employed.

Executive summary:

The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

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

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 34 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution (3 replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test material solutions were prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test material. 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 3.1 to 33 mg/L. A decline in measured test concentrations after each 24-Hour period was observed in the range of 0.27 to 30 mg/L at 24 hours, 0.010 to 14 mg/L at 48 hours and from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0016 mg/L, to 0.015 mg/L at 72 hours. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. These were determined to be 0.32, 0.35, 3.0, 5.8 and 17 mg/L.

It was not possible to calculate ErC20 or ErC50 values as no concentration tested resulted in greater than 20 % inhibition of growth rate. The EyC10 value was determined by extrapolation of the curve as at the lowest test concentration employed, 16 % inhibition of yield occurred. It was not possible to calculate an EyC50 value as no concentration tested resulted in greater than 44 % inhibition of yield.

Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the geometric mean measured test concentrations: Growth rate and yield inhibition EC50 > 17 mg/L, growth rate NOEC of 3.0 mg/L and growth rate LOEC of 5.8 mg/L.

Description of key information

The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 34 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution (3 replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test material solutions were prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test material. 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 3.1 to 33 mg/L. A decline in measured test concentrations after each 24-Hour period was observed in the range of 0.27 to 30 mg/L at 24 hours, 0.010 to 14 mg/L at 48 hours and from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0016 mg/L, to 0.015 mg/L at 72 hours. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. These were determined to be 0.32, 0.35, 3.0, 5.8 and 17 mg/L.

It was not possible to calculate ErC20 or ErC50 values as no concentration tested resulted in greater than 20 % inhibition of growth rate. The EyC10 value was determined by extrapolation of the curve as at the lowest test concentration employed, 16 % inhibition of yield occurred. It was not possible to calculate an EyC50 value as no concentration tested resulted in greater than 44 % inhibition of yield.

Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the geometric mean measured test concentrations: Growth rate and yield inhibition EC50 > 17 mg/L, growth rate NOEC of 3.0 mg/L and growth rate LOEC of 5.8 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

3 mg/L