1-Propanaminium, N,N,N-trimethyl-3-(octadecyloxy)-, chloride (1:1)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 July 2007 to 5 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21 August 2007 Date of Signature: 15 October 2007

Test material

Test material information

Constituent 1

Reference substance name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

EC Number:

700-414-8

Cas Number:

23328-71-4

Molecular formula:

C24H52ClNO

IUPAC Name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

Details on test material:

- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Exp. I-070518
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: The test samples were tested for stability without prior mixing (sonication) of the test sample bottles to assess for losses due to adsorption and/or insolubility.
The bottom level light and dark and middle level dark samples were just above the normal acceptance limit of 120%. However as most of the other values were just below 120% the difference is not considered significant and the test samples have been considered to be stable in the test medium.
The 0.10 mg/l unsonicated stability vessel showed evidence of insolubility or adherence to glass.
- Storage condition of test material: room temperature in the dark
- Other: Not reported.

Specific details on test material used for the study:

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored at approximately 20ºC for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
For the purpose of the definitive test, the test material was dissolved directly in culture medium.
An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 20, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/l. An aliquot (500 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (500 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

- Eluate:
Not applicable

- Differential loading:
Not applicable

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

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Not applicable

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Green Algae

- Strain:
CCAP 276/20

- Source (laboratory, culture collection):
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 (at test initiation):
Not recorded

- Method of cultivation:
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 deg C.

ACCLIMATION
- Acclimation period:
Not applicable

- Culturing media and conditions (same as test or not):
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

- Any deformed or abnormal cells observed:
None observed.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

Not recorded.

Test temperature:

Temperature within the incubator was recorded daily.

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

pH:

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.

The pH values of the control cultures (see Table 4) were observed to increase from pH 7.4 at 0 hours to pH 7.6 – 7.7 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.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The initial range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.

A second range-finding test was conducted by exposing Desmodesmus subspicatus cells to nominal test concentrations of 0.00010, 0.0010, 0.010, 0.10 and 1.0 mg/l for a period of 72 hours.

Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Please see Table 3 for measured concentrations found.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 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.
- Aeration: Not aerated

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

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

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 5.04 x 105 cells per ml. This suspension was diluted to a cell density of 8.34 x 103 cells per ml prior to use. At initiation of the test the culture contained a nominal cell density of 4 x 103 cells per ml.

- Control end cells density:
Mean cell density of control at 72 hours : 1.42x 105 cells per ml

- No. of organisms per vessel:
Not applicable

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

- No. of vessels per control (replicates):
4 replicates.

- No. of vessels per vehicle control (replicates):
Not applicable

GROWTH MEDIUM
- Standard medium used: yes

- Detailed composition if non-standard medium was used:
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* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
* Elga Optima 15+ or Elga Purelab Option R-15 BP

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
reverse osmosis purified deionised water, Elga Optima 15+ or Elga Purelab Option R-15 BP

- Total organic carbon:
Not recorded

- Particulate matter:
Not recorded

- Metals:
Not recorded

- Pesticides:
Not recorded

- Chlorine:
Not recorded

- Alkalinity:
Same as test conditions

- Ca/mg ratio:
Not recorded

- Conductivity:
Not recorded

- Culture medium different from 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.

- Intervals of water quality measurement:
Not recorded

OTHER TEST CONDITIONS
- Sterile test conditions: yes

- Adjustment of pH:
No

- Photoperiod:
continuous illumination

- Light intensity and quality:
intensity approximately 7000 lux provided by warm white lighting (380 – 730 nm)

- Salinity (for marine algae):
Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
Samples were taken at 0, 24, 51 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

- Chlorophyll measurement:
Not applicable

-Other:
Evaluation of data
- Comparison of growth rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

u = 1n N – 1n N1 / tn – t1

where:
u = 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.

Ir = uc – ut / uc x 100

Percentage inhibition of growth rate for each replicate test material vessel was calculated using the following equation:

where:
Ir = percentage inhibition of average specific growth rate
uc = mean average specific growth rate for the control cultures
ut = 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

Comparison of biomass integral
The biomass integral (area under the growth curve) was calculated using the following equation:

A = N1 – N0 / 2 x t1 + N1 + N2 -2N0 / 2 x (t2 – t1) + Nn-1 + Nn – 2N0 / 2 x (tn – tn-1)

where:
A = area
N0 = nominal cell concentration at start of test
N1 = measured cell concentration at t1
Nn = measured cell concentration at tn
t1 = time of first measurement after beginning of test
tn = time of nth measurement after beginning of test

Percentage inhibition of the biomass integral for each replicate test material vessel was calculated using the following equation:

IA = Ac – At / Ac x 100

where:
IA = percentage inhibition of the biomass integral
Ac = mean biomass integral for the control cultures
At = biomass integral for the test culture

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). 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).

Geometric mean measured test concentrations
The geometric mean measured test concentrations of the samples were calculated as follows using the measured test concentrations of replicates R1 - R3 pooled:

GM = (C0 x C1) sq root

where
GM = geometric mean measured test concentration (mg/l)
C0 = measured concentration at the start of the test (mg/l)
C1 = measured concentration at the end of the test (mg/l)


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
initial range-finding: 0.10, 1.0, 10 and 100 mg/l
second range-finding: 0.00010, 0.0010, 0.010, 0.10 and 1.0 mg/l
definitive test: 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

- Justification for using less concentrations than requested by guideline:
The test concentrations to be used in the definitive test were determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Desmodesmus subspicatus 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 containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks, each containing 100 ml of test preparation were used for each control and test concentration. The test material was dissolved directly in culture medium.

An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 20, 2.0 and 0.20 mg/l. An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 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 results of the initial range-finding test showed that significant inhibition of growth occurred at all test concentrations employed and so a second range-finding test was conducted by exposing Desmodesmus subspicatus cells to nominal test concentrations of 0.00010, 0.0010, 0.010, 0.10 and 1.0 mg/l for a period of 72 hours.

An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 20, 2.0, 0.20, 0.020, 0.0020 and 0.00020 mg/l. An aliquot (100 ml) of each of the 0.00020, 0.0020, 0.020, 0.20 and 2.0 mg/l stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 0.00010, 0.0010, 0.010, 0.10 and 10 mg/l.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The results obtained for the 0.10 mg/l test concentration in the second range-finding test were inconsistent with those obtained from the initial range-finding test conducted and therefore a third range-finding test was conducted at concentrations of 0.010, 0.10 and 1.0 mg/l in order to confirm the results obtained.

An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 20, 2.0, 0.20 and 0.020 mg/l. An aliquot (100 ml) of each of the 0.020, 0.20 and 2.0 mg/l stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/l.


- Range finding study
- Test concentrations:
As above
- Results used to determine the conditions for the definitive study:
Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

Validation Criteria
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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.078 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 0.067 – 0.092 mg/l

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.032 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits 0.029 – 0.035 mg/l

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.031 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits 0.027 – 0.035 mg/l

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: 95% CL not recorded

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: 95% CL not recorded

Details on results:

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

- Observation of abnormalities (for algal test):
There were no abnormalities detected in any of the control or test cultures at 72 hours.

- Any stimulation of growth found in any treatment:
The following data show that the cell concentration of the control cultures increased by a factor of 38 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

The mean coefficient of variation for section by section specific growth rate for the control cultures was 25% 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 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 40% to 60% of nominal. These low measured test concentrations were considered to be due to possible absorption to the glassware used in the preparation of the test series, given that the test material was a quaternary ammonium compound. Whilst in hindsight the use of pre-conditioned glassware may have overcome the low 0-Hour measured concentrations, given that a significant toxic response was observed at the measured test concentrations employed this was considered to have had no adverse effect on the outcome of the test.

Analysis of the test preparations at 72 hours showed a decline in measured concentrations in the range of 12% to 43% of nominal. This decline was considered to be due to a combination of absorption to glassware and adsorption to the algal cells present. Whilst the recovery analyses conducted in the presence of algal cells indicated that no significant absorption occurred this does not preclude long-term adsorption over the test period.

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.

- Effect concentrations exceeding solubility of substance in test medium:
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, 0.0625 and 0.125 mg/l test cultures were observed to be pale green dispersions. The 0.25 mg/l test cultures were very pale green dispersions, the 0.50 mg/l test cultures were extremely pale green dispersions and the 1.0 mg/l test cultures were clear colourless solutions.

Results with reference substance (positive control):

The cell densities from exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material during the positive control (Safepharm Laboratories Project No: 0039/0941) are given in Table 7. Daily specific growth rates for the control cultures are given in Table 8 whilst growth rates, yield and biomass integral values are given in Table 9.

Accordingly the following results were determined from the data:

ErC50 (0 - 72 h) : 0.49 mg/l; 95% confidence limits 0.43 - 0.55 mg/l
EyC50 (0 - 72 h) : 0.22 mg/l; 95% confidence limits 0.19 - 0.24 mg/l
EbC50 (0 - 72 h) : 0.23 mg/l; 95% confidence limits 0.21 - 0.27 mg/l

No Observed Effect Concentration (NOEC) based on growth rate : 0.125 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral : 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on biomass integral : 0.125 mg/l

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

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, yield and biomass integral 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).

Any other information on results incl. tables

Table 1 Cell Densities and Percentage Inhibition of Growth from the Initial Range-finding Test

Nominal Concentration		Cell Densities* (cells per ml)		Inhibition Values (%)
(mg/l)		0 Hours	72 Hours	Growth Rate	Yield/Biomass Integral
Control	R1	4.92E+03	2.76E+05
	R2	3.80E+03	2.54E+05	-	-
	Mean	4.36E+03	2.65E+05
0.1	R1	4.13E+03	1.59E+05
	R2	4.26E+03	1.17E+05	16	49
	Mean	4.19E+03	1.38E+05
1	R1	4.77E+03	7.77E+03
	R2	4.42E+03	5.19E+03	91	99
	Mean	4.59E+03	6.48E+03
10	R1	4.21E+03	6.62E+03
	R2	4.27E+03	6.51E+03	89	99
	Mean	4.24E+03	6.56E+03
100	R1	4.17E+03	4.25E+03
	R2	4.52E+03	5.14E+03	98	100
	Mean	4.35E+03	4.69E+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 2

Table 2 Cell Densities and Percentage Inhibition of Growth from the Second Range-finding Test

Nominal Concentration		Cell Densities* (cells per ml)		Inhibition Values (%)
(mg/l)		0 Hours	72 Hours	Growth Rate	Yield/Biomass Integral
Control	R1	4.07E+03	4.41E+05
	R2	4.20E+03	4.05E+05	-	-
	Mean	4.14E+03	4.23E+05
0.0001	R1	4.50E+03	5.65E+05
	R2	4.73E+03	3.82E+05	0	[12]
	Mean	4.62E+03	4.73E+05
0.001	R1	4.92E+03	4.37E+05
	R2	4.19E+03	4.63E+05	0	[6]
	Mean	4.56E+03	4.50E+05
0.01	R1	4.25E+03	6.47E+05
	R2	4.35E+03	6.31E+05	[8]	[52]
	Mean	4.30E+03	6.39E+05
0.1	R1	4.52E+03	7.15E+05
	R2	4.63E+03	7.30E+05	[9]	[71]
	Mean	4.58E+03	7.23E+05
1	R1	4.58E+03	3.63E+04
	R2	4.72E+03	2.59E+04	59	94
	Mean	4.65E+03	3.11E+04

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.R₁and R₂= Replicates 1 and 2

Table 3 Cell Densities and Percentage Inhibition of Growth from the Third Range-finding Test

Nominal Concentration		Cell Densities* (cells per ml)		Inhibition Values (%)
(mg/l)		0 Hours	72 Hours	Growth Rate	Yield/Biomass Integral
Control	R1	4.00E+03	4.01E+05
	R2	3.98E+03	3.13E+05	-	-
	Mean	3.99E+03	3.57E+05
0.01	R1	4.08E+03	3.70E+05
	R2	4.07E+03	3.41E+05	0	0
	Mean	4.07E+03	3.55E+05
0.1	R1	3.96E+03	2.95E+05
	R2	4.04E+03	3.42E+05	2	11
	Mean	4.00E+03	3.18E+05
1	R1	4.32E+03	5.04E+03
	R2	4.28E+03	6.06E+03	94	100
	Mean	4.30E+03	5.55E+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 2

[Increase in growth compared to controls] Table 4 Cell Densities and pH Values in the Definitive Test

Nominal Concentration		pH	Cell Densities* (cells per ml)				pH
(mg/l)		0 h	0 h	24 h	51 h	72 h	72 h
Control	R1	7.4	3.34E+03	1.01E+04	5.77E+04	1.52E+05	7.6
	R2	7.4	3.67E+03	9.14E+03	5.61E+04	1.45E+05	7.7
	R3	7.4	3.58E+03	9.35E+03	4.20E+04	1.47E+05	7.6
	R4	7.4	2.87E+03	1.17E+04	5.39E+04	1.30E+05	7.6
	R5	7.4	3.92E+03	1.06E+04	5.58E+04	1.39E+05	7.6
	R6	7.4	4.83E+03	1.01E+04	5.47E+04	1.41E+05	7.6
	Mean		3.70E+03	1.02E+04	5.34E+04	1.42E+05
0.0625	R1	7.4	3.54E+03	1.06E+04	2.56E+04	1.40E+05	7.6
	R2	7.4	2.84E+03	1.05E+04	5.42E+04	1.39E+05	7.6
	R3	7.4	3.94E+03	1.08E+04	5.71E+04	1.61E+05	7.5
	Mean		3.44E+03	1.06E+04	4.56E+04	1.47E+05
0.125	R1	7.3	3.94E+03	1.18E+04	1.37E+04	7.29E+04	7.5
	R2	7.3	4.08E+03	1.19E+04	2.50E+04	7.62E+04	7.5
	R3	7.3	3.78E+03	1.20E+04	2.02E+04	8.78E+04	7.5
	Mean		3.93E+03	1.19E+04	1.97E+04	7.90E+04
0.25	R1	7.3	3.53E+03	6.24E+03	2.89E+04	2.53E+04	7.4
	R2	7.3	3.94E+03	6.41E+03	2.82E+04	2.61E+04	7.4
	R3	7.3	4.08E+03	7.23E+03	2.65E+04	3.20E+04	7.4
	Mean		3.85E+03	6.63E+03	2.79E+04	2.78E+04
0.5	R1	7.3	4.33E+03	4.01E+03	7.84E+03	1.67E+04	7.3
	R2	7.3	4.60E+03	3.91E+03	8.40E+03	1.40E+04	7.3
	R3	7.3	4.24E+03	5.27E+03	7.98E+03	1.02E+04	7.3
	Mean		4.39E+03	4.39E+03	8.08E+03	1.36E+04
1	R1	7.3	4.51E+03	1.62E+03	4.22E+03	5.62E+03	7.3
	R2	7.3	4.87E+03	1.37E+03	4.38E+03	8.30E+03	7.3
	R3	7.3	4.30E+03	2.23E+03	3.85E+03	4.55E+03	7.3
	Mean		4.56E+03	1.74E+03	4.15E+03	6.15E+03

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

R₁- R₆= Replicates 1 to 6

Table 5 Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.039	0.065	0.046
	R2	0.034	0.067	0.045
	R3	0.035	0.056	0.06
	R4	0.045	0.057	0.042
	R5	0.041	0.061	0.044
	R6	0.039	0.062	0.045
	Mean	0.039	0.061	0.047

R₁- R₆= Replicates 1 to 6

Table 6 Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test

Nominal Concentration		Growth Rate		Yield		Biomass Integral
(mg/l)		(cells/ml/hour)		(cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*	0 – 72 h	% Inhibition
Control	R1	0.05		1.48E+05		2.99E+06
	R2	0.05		1.41E+05		2.86E+06
	R3	0.05		1.44E+05		2.56E+06
	R4	0.048	-	1.27E+05	-	2.72E+06	-
	R5	0.049		1.36E+05		2.83E+06
	R6	0.049		1.36E+05		2.81E+06
	Mean	0.049		1.39E+05		2.80E+06
	SD	0.001		7.37E+03		1.48E+05
0.0625	R1	0.049	0	1.37E+05		2.12E+06	24
	R2	0.049	0	1.36E+05		2.79E+06	0
	R3	0.051	[4]	1.57E+05		3.10E+06	[11]
	Mean	0.05	[1]	1.43E+05	[3]	2.67E+06	4
	SD	0.001		1.21E+04		5.02E+05
0.125	R1	0.04	18	6.90E+04		1.16E+06	59
	R2	0.041	16	7.22E+04		1.46E+06	48
	R3	0.043	12	8.40E+04		1.47E+06	47
	Mean	0.041	15	7.51E+04	46	1.36E+06	51
	SD	0.002		7.94E+03		1.82E+05
0.25	R1	0.026	47	2.18E+04		8.79E+05	69
	R2	0.026	47	2.22E+04		8.74E+05	69
	R3	0.029	41	2.79E+04		9.17E+05	67
	Mean	0.027	45	2.40E+04	83	8.90E+05	68
	SD	0.002		3.42E+03		2.36E+04
0.5	R1	0.02	59	1.24E+04		2.26E+05	92
	R2	0.017	65	9.41E+03		2.08E+05	93
	R3	0.013	73	5.93E+03		1.93E+05	93
	Mean	0.017	66	9.24E+03	93	2.09E+05	93
	SD	0.004		3.23E+03		1.67E+04
1	R1	0.005	90	1.10E+03		-3.86E+04	101
	R2	0.01	80	3.43E+03		-1.29E+04	100
	R3	0.002	96	2.52E+02		-4.29E+04	102
	Mean	0.006	89	1.59E+03	99	-3.15E+04	101
	SD	0.004		1.64E+03		1.63E+04

*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] Table 7 Cell Densities and pH Values in the Positive Control

Nominal Concentration		pH	Cell Densities* (cells per ml)				pH
(mg/l)		0 h	0 h	28 h	52 h	72 h	72 h
Control	R1	7.3	4.17E+03	1.70E+04	9.72E+04	5.36E+05	7.7
	R2	7.3	4.36E+03	1.69E+04	1.07E+05	4.72E+05	7.7
	R3	7.3	4.23E+03	1.73E+04	8.95E+04	4.34E+05	7.7
	R4	7.3	4.26E+03	1.75E+04	1.18E+05	5.19E+05	7.8
	R5	7.3	4.14E+03	1.79E+04	1.07E+05	4.90E+05	7.8
	R6	7.3	4.08E+03	1.65E+04	7.85E+04	4.63E+05	7.7
	Mean		4.20E+03	1.72E+04	9.96E+04	4.86E+05
0.0625	R1	7.3	4.22E+03	2.04E+04	8.90E+04	5.69E+05	7.7
	R2	7.3	4.15E+03	1.87E+04	9.39E+04	4.95E+05	7.7
	R3	7.3	4.28E+03	1.64E+04	9.56E+04	5.29E+05	7.7
	Mean		4.22E+03	1.85E+04	9.28E+04	5.31E+05
0.125	R1	7.3	3.88E+03	1.51E+04	9.00E+04	4.07E+05	7.8
	R2	7.3	4.11E+03	1.58E+04	9.07E+04	3.56E+05	7.7
	R3	7.3	4.27E+03	1.64E+04	9.34E+04	3.70E+05	7.7
	Mean		4.09E+03	1.57E+04	9.13E+04	3.78E+05
0.25	R1	7.3	4.08E+03	1.01E+04	6.46E+04	1.98E+05	7.7
	R2	7.3	4.03E+03	1.02E+04	5.37E+04	2.11E+05	7.7
	R3	7.3	4.28E+03	1.08E+04	3.98E+04	2.14E+05	7.7
	Mean		4.13E+03	1.04E+04	5.27E+04	2.08E+05
0.5	R1	7.3	4.32E+03	9.58E+03	2.19E+04	5.41E+04	7.6
	R2	7.3	4.48E+03	1.20E+04	2.92E+04	4.46E+04	7.6
	R3	7.3	4.24E+03	1.06E+04	2.01E+04	2.78E+04	7.6
	Mean		4.35E+03	1.07E+04	2.37E+04	4.22E+04
1	R1	7.2	4.10E+03	8.28E+03	8.79E+03	1.15E+04	7.5
	R2	7.2	4.12E+03	7.62E+03	6.37E+03	9.70E+03	7.5
	R3	7.2	4.01E+03	6.97E+03	5.69E+03	8.80E+03	7.5
	Mean		4.07E+03	7.62E+03	6.95E+03	9.99E+03

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

R₁- R₆= Replicates 1 to 6 Table 8 Daily Specific Growth Rates for the Control Cultures in the Positive Control

		Daily Specific Growth Rate
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.052	0.073	0.085
	R2	0.051	0.077	0.074
	R3	0.052	0.068	0.079
	R4	0.053	0.08	0.074
	R5	0.054	0.074	0.076
	R6	0.051	0.065	0.089
	Mean	0.052	0.073	0.08

R₁- R₆= Replicates 1 to 6

Table 9 Inhibition of Growth Rate, Yield and Biomass Integral in the Positive Control

Nominal Concentration		Growth Rate		Yield		Biomass Integral
(mg/l)		(cells/ml/hour)		(cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*	0 – 72 h	% Inhibition
Control	R1	0.068		5.32E+05		7.71E+06
	R2	0.066		4.68E+05		7.29E+06
	R3	0.065		4.30E+05		6.53E+06
	R4	0.068	-	5.15E+05	-	8.02E+06	-
	R5	0.067		4.86E+05		7.49E+06
	R6	0.066		4.59E+05		6.55E+06
	Mean	0.067		4.82E+05		7.27E+06
	SD	0.001		3.75E+04		6.09E+05
0.0625	R1	0.069	[3]	5.65E+05		7.95E+06	[9]
	R2	0.067	0	4.91E+05		7.27E+06	0
	R3	0.068	[1]	5.24E+05		7.58E+06	[4]
	Mean	0.068	[1]	5.27E+05	[9]	7.60E+06	[4]
	SD	0.001		3.70E+04		3.37E+05
0.125	R1	0.064	4	4.03E+05		6.21E+06	15
	R2	0.062	7	3.52E+05		5.73E+06	21
	R3	0.063	6	3.65E+05		5.95E+06	18
	Mean	0.063	6	3.73E+05	22	5.96E+06	18
	SD	0.001		2.68E+04		2.41E+05
0.25	R1	0.054	19	1.94E+05		3.43E+06	53
	R2	0.055	18	2.07E+05		3.32E+06	54
	R3	0.055	18	2.10E+05		3.07E+06	58
	Mean	0.055	18	2.04E+05	58	3.28E+06	55
	SD	0.001		8.43E+03		1.87E+05
0.5	R1	0.036	46	4.97E+04		1.04E+06	86
	R2	0.033	51	4.01E+04		1.17E+06	84
	R3	0.027	60	2.36E+04		7.65E+05	89
	Mean	0.032	52	3.78E+04	92	9.91E+05	86
	SD	0.005		1.32E+04		2.06E+05
1	R1	0.015	78	7.37E+03		2.91E+05	96
	R2	0.012	82	5.58E+03		2.03E+05	97
	R3	0.011	84	4.79E+03		1.62E+05	98
	Mean	0.013	81	5.91E+03	99	2.19E+05	97
	SD	0.002		1.32E+03		6.60E+04

*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]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period. Based on the geometric mean measured test concentrations the ErC50 (0 - 72 h) value was 0.078 mg/l; 95% confidence limits 0.067 – 0.092 mg/l, the EyC50 (0 - 72 h) value was 0.032 mg/l; 95% confidence limits 0.029 – 0.035 mg/l and the EbC50 (0 - 72 h) value was 0.031 mg/l; 95% confidence limits 0.027 – 0.035 mg/l. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 0.028 mg/l, and the No Observed Effect Concentration was 0.017 mg/l.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. 

Following preliminary range-finding tests, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

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

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatuswas exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

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

Results.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 40% to 60% of nominal. These low measured test concentrations were considered to be due to possible absorption to the glassware used in the preparation of the test series, given that the test material was a quaternary ammonium compound.

Analysis of the test preparations at 72 hours showed a decline in measured concentrations in the range of 12% to 43% of nominal. This decline was considered to be due to a combination of absorption to glassware and adsorption to the algal cells present. Whilst the recovery analyses conducted in the presence of algal cells indicated that no significant absorption occurred this does not preclude long-term adsorption over the test period. 

Given the low 0-Hour measured test concentrations and the decline in measured test concentrations over the test period it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The E_rC₅₀(0 - 72 h) based on the geometric mean measured test concentrations was 0.078 mg/l; 95% confidence limits 0.067 – 0.092 mg/l, the E_yC₅₀(0 - 72 h) was 0.032 mg/l; 95% confidence limits 0.029 – 0.035 mg/l, and the E_bC₅₀(0 - 72 h) was 0.031 mg/l; 95% confidence limits    0.027 – 0.035 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral was 0.028 mg/l and the No Observed Effect Concentration was 0.017 mg/l.

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an E_rC₅₀(0 - 72 h) of 0.49 mg/l; 95% confidence limits 0.43 – 0.55 mg/l, an E_yC₅₀(0 - 72 h) of 0.22 mg/l; 95% confidence limits 0.19 ‑ 0.24 mg/l, and an E_bC₅₀(0 - 72 h) of 0.23 mg/l; 95% confidence limits 0.21 - 0.27 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral were 0.25, 0.125 and 0.125 mg/l respectively and the No Observed Effect Concentrations were 0.125, 0.0625 and 0.0625 mg/l respectively.