Diallylamine

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 18 August 2015 and 04 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours .

Vehicle:

no

Details on test solutions:

Range- Finding Test
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 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers to reduce evaporation and losses through volatilization. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (6.9 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
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 sealed with ground glass stoppers 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 immediate chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation
Nominal amounts of test item (100 and 32 mg) were each separately dissolved in culture medium and the volume adjusted to 1 liter to give 100 and 32 mg/L stock solutions respectively from which a series of dilutions was made to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (8.3 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
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 and 72 hours

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 ºC. initial cell density was approximately 10x3 cells/mL.

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.

Culture Medium : The media used in both the range-finding and definitive tests was prepared with the addition of 500 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system.

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
Na2EDTA.2H2O 0.30 mg/L

The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

No data

Test temperature:

The temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH value of the control cultures 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.

Dissolved oxygen:

No data

Salinity:

Not applicable as freshwater used

Nominal and measured concentrations:

Following a preliminary range-finding test,the nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were used. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.67 to 80 mg/L (67% to 80% of the nominal test concentrations). Concentrations in the range of 0.73 to 84 mg/L (73% to 87% of the nominal test concentrations) were obtained at 72 hours.

Details on test conditions:

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 item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.39 x 105 cells per mL. Inoculation of 900 mL of test medium with 8.3 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 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.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined 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 computerized interpolation using the Xlfit software package (IDBS). ECx values 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).

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 daily 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:

EC50

Effect conc.:

28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 22-35 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 7.5-12 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 22-35 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 7.5-12 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 22-35 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 7.5-12 mg/L

Details on results:

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1. The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 85% to 101% of nominal indicating that the test item was stable over the test duration.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.67 to 80 mg/L (67% to 80% of the nominal test concentrations). Concentrations in the range of 0.73 to 84 mg/L (73% to 87% of the nominal test concentrations) were obtained at 72 hours. It was considered that the failure to obtain near nominal concentrations in all test samples at both 0 and 72 hours was due to losses during the analytical work up process despite all samples being taken for immediate analysis.

Given this it was considered justifiable to base the results on the geometric mean measured test concentrations in accordance with current test guidelines.

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration Geometric Mean Measured Test Concentration (mg/L) Expressed as a % of the Nominal Test Concentration
(mg/L)
1.0 0.70 70
3.2 2.6 81
10 8.2 82
32 26 81
100 82 82

Growth Data
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. From the data given in Table 2 and Table 4, 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 over the 72-Hour exposure period.

Accordingly the following results were determined from the data based on the geometric mean measured test concentrations:
Inhibition of Growth Rate
ErC10 (0 - 72 h) : 9.3 mg/L
ErC20 (0 - 72 h) : 14 mg/L
ErC50 (0 - 72 h) : 28 mg/L; 95% confidence limits 22 - 35 mg/L

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

Inhibition of Yield
EyC10 (0 - 72 h) : 2.0 mg/L
EyC20 (0 - 72 h) : 3.5 mg/L
EyC50 (0 - 72 h) : 9.4 mg/L; 95% confidence limits 7.5 - 12 mg/L

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

Validation Criteria:
The following data show that the cell concentration of the control cultures increased by a factor of 81 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 : 6.20 x 103 cells per mL
Mean cell density of control at 72 hours : 5.03 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 26% 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%.

Observation 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 0.70, 2.6 and 8.2 mg/L, however no intact cells were observed to be present in the test cultures at 26 and 82 mg/L.

Water Quality Criteria
The temperature was maintained at 24 ± 1 ºC throughout the test. The pH value of the control cultures was observed to increase from pH 8.1 at 0 hours to pH 9.2 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.

There was a concentration dependant increase in pH at 0 hours in the range of pH 8.1 at 0.70 mg/L through to pH 9.3 at 82 mg/L. This was considered to be due to an intrinsic property of the test item and as such no adjustments to the pH were made. Furthermore, effects on growth were observed at the lower test concentrations employed where the pH did not differ significantly from that of the control and as such it was considered unlikely that it was the pH of the test media which was causing the toxicity observed.

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, 0.70, 2.6 and 8.2 mg/L test cultures were observed to be green dispersions whilst the 26 and 82 mg/l test cultures were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control (Study Number 41501180) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 12 May 2015 and 15 May 2015.
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) : 1.0 mg/L; 95% confidence limits 0.90 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.49 mg/L; 95% confidence limits 0.42 – 0.58 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:

Statistical analysis of the growth rate data was carried out for the control 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 (P0.05), between the control and 0.70 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.70 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 2.6 mg/L.

Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences (P0.05), between the control and 0.70 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.70 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 2.6 mg/L.

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

Nominal Concentration (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.78E+03	3.38E+05	-	-
	R2	6.42E+03	2.22E+05
	Mean	6.60E+03	2.80E+05
0.10	R1	6.86E+03	2.90E+05	[8]	[20]
	R2	5.07E+03	3.76E+05
	Mean	5.97E+03	3.33E+05
1.0	R1	7.19E+03	2.43E+05	2	12
	R2	5.31E+03	2.50E+05
	Mean	6.25E+03	2.47E+05
10	R1	5.69E+03	2.75E+05	0	7
	R2	6.34E+03	2.45E+05
	Mean	6.01E+03	2.60E+05
100	R1	5.95E+03	1.07E+04	90	99
	R2	6.37E+03	6.81E+03
	Mean	6.16E+03	8.77E+03

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

R₁and R₂= Replicates 1 and

[Increase in growth compared to controls]

Table2     Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.1	6.16E+03	1.53E+04	1.02E+05	4.16E+05	9.2
	R2		5.37E+03	1.80E+04	8.23E+04	5.70E+05
	R3		6.31E+03	1.54E+04	6.75E+04	5.26E+05
	R4		6.78E+03	1.72E+04	6.44E+04	5.26E+05
	R5		6.45E+03	1.77E+04	1.26E+05	4.66E+05
	R6		6.16E+03	1.66E+04	1.03E+05	5.13E+05
	Mean		6.20E+03	1.67E+04	9.08E+04	5.03E+05
0.70	R1	8.1	5.72E+03	1.56E+04	8.39E+04	4.55E+05	9.2
	R2		6.25E+03	1.56E+04	7.66E+04	5.16E+05
	R3		6.95E+03	7.66E+03	6.10E+04	5.22E+05
	Mean		6.31E+03	1.30E+04	7.39E+04	4.98E+05
2.6	R1	8.1	6.25E+03	1.49E+04	9.28E+04	3.25E+05	9.2
	R2		6.63E+03	1.60E+04	6.95E+04	3.95E+05
	R3		6.45E+03	1.15E+04	9.06E+04	3.62E+05
	Mean		6.44E+03	1.41E+04	8.43E+04	3.61E+05
8.2	R1	8.1	5.72E+03	1.55E+04	8.29E+04	3.36E+05	9.1
	R2		5.46E+03	1.55E+04	3.64E+04	3.50E+05
	R3		5.84E+03	1.50E+04	7.28E+04	3.51E+05
	Mean		5.67E+03	1.54E+04	6.40E+04	3.46E+05
26	R1	8.5	5.84E+03	5.22E+03	1.22E+04	5.96E+04	8.7
	R2		9.12E+03	4.72E+03	8.45E+03	6.15E+04
	R3		6.10E+03	4.78E+03	1.24E+04	6.36E+04
	Mean		7.02E+03	4.91E+03	1.10E+04	6.16E+04
82	R1	9.3	6.89E+03	4.08E+03	4.02E+03	7.95E+03	9.2
	R2		7.95E+03	3.29E+03	3.40E+03	6.04E+03
	R3		7.51E+03	3.87E+03	2.70E+03	8.86E+03
	Mean		7.45E+03	3.74E+03	3.37E+03	7.62E+03

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

R₁- R₆= Replicates 1 to 6

Table3     Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.047	0.079	0.059
	R2	0.053	0.063	0.081
	R3	0.047	0.062	0.086
	R4	0.051	0.055	0.088
	R5	0.053	0.082	0.054
	R6	0.050	0.076	0.067
	Mean	0.050	0.070	0.073

R₁- R₆= Replicates 1 to 6

Table4     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 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.061		4.10E+05
	R2	0.066		5.65E+05
	R3	0.065		5.19E+05
	R4	0.065	-	5.19E+05	-
	R5	0.063		4.59E+05
	R6	0.064		5.07E+05
	Mean	0.064		4.97E+05
	SD	0.002		5.43E+04
0.70	R1	0.063	2	4.49E+05
	R2	0.064	0	5.09E+05
	R3	0.065	[2]	5.15E+05
	Mean	0.064	0	4.91E+05	1
	SD	0.001		3.63E+04
2.6	R1	0.058	9	3.19E+05
	R2	0.061	5	3.89E+05
	R3	0.059	8	3.56E+05
	Mean	0.059	7	3.54E+05	29
	SD	0.002		3.51E+04
8.2	R1	0.058	9	3.30E+05
	R2	0.059	8	3.44E+05
	R3	0.059	8	3.45E+05
	Mean	0.059	8	3.40E+05	32
	SD	0.001		8.33E+03
26	R1	0.034	47	5.38E+04
	R2	0.035	45	5.24E+04
	R3	0.035	45	5.75E+04
	Mean	0.035	46	5.46E+04	89
	SD	0.001		2.64E+03
82	R1	0.006	91	1.06E+03
	R2	0.003	95	-1.91E+03
	R3	0.008	88	1.35E+03
	Mean	0.006	91	1.66E+02	100
	SD	0.003		1.80E+03

*In accordance with the OECD test guideline only thean 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]

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:
Response Variable EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate 28 22-35 0.70 2.6
Yield 9.4 7.5-12 0.70 2.6

Executive summary:

Introduction

A study was perford to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. The method followed was designed to be compatible with 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……..

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Hermanet al1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

 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 test preparations at 0 hours showed measured test concentrations to range from 0.67 to 80 mg/L (67% to 80% of the nominal test concentrations). Concentrations in the range of 0.73 to 84 mg/L (73% to 87% of the nominal test concentrations) were obtained at 72 hours. It was considered that the failure to obtain near nominal concentrations in all test samples at both 0 and 72 hours was due to losses during the analytical work up process despite all samples being taken for immediate analysis.

 Given this it was considered justifiable to base the results on the geometric mean measured test concentrations.

 

Exposure ofPseudokirchneriella subcapitatato the test item gave the following results based on the geometric mean measured test concentrations:

 

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	28	22	-	35	0.70	2.6
Yield	9.4	7.5	-	12	0.70	2.6

 

 

Description of key information

Introduction

A study was perford to assess the effect of the test item on the growth of the green alga Pseudokirchneriellasubcapitata. The method followed was designed to be compatible with 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……..

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman etc al 1990) in order to prevent inhibition of growth due to the restrictions of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth..

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 test preparations at 0 hours showed measured test concentrations to range from 0.67 to 80 mg/L (67% to 80% of the nominal test concentrations). Concentrations in the range of 0.73 to 84 mg/L (73% to 87% of the nominal test concentrations) were obtained at 72 hours. It was considered that the failure to obtain near nominal concentrations in all test samples at both 0 and 72 hours was due to losses during the analytical work up process despite all samples being taken for immediate analysis.

Given this it was considered justifiable to base the results on the geometric mean measured test concentrations.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	28	22	-	35	0.70	2.6
Yield	9.4	7.5	-	12	0.70	2.6

Key value for chemical safety assessment

EC50 for freshwater algae:

28 mg/L

EC10 or NOEC for freshwater algae:

0.7 mg/L