1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether

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:

This study was conducted between 11 September 2017 and 12 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EC No. 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-​Propanol, 1-​[[[3-​(aminomethyl)​phenyl]​methyl]​amino]​-​3-​(2-​methylphenoxy)​-

Cas Number:

1617528-47-8

Molecular formula:

C18H24N2O2

IUPAC Name:

2-​Propanol, 1-​[[[3-​(aminomethyl)​phenyl]​methyl]​amino]​-​3-​(2-​methylphenoxy)​-

Constituent 2

Reference substance name:

3,3'-[benzene-1,3-diylbis(methanediylimino)]bis[1-(2-methylphenoxy)propan-2-ol]

Molecular formula:

C28H36N2O4

IUPAC Name:

3,3'-[benzene-1,3-diylbis(methanediylimino)]bis[1-(2-methylphenoxy)propan-2-ol]

Constituent 3

Reference substance name:

m-phenylenebis(methylamine)

EC Number:

216-032-5

EC Name:

m-phenylenebis(methylamine)

Cas Number:

1477-55-0

Molecular formula:

C8H12N2

IUPAC Name:

1,3-phenylenedimethanamine

impurity 1

Reference substance name:

2-Propanol, 1,3-bis(2-methylphenoxy)-

Cas Number:

17181-49-6

Molecular formula:

C17H20O3

IUPAC Name:

2-Propanol, 1,3-bis(2-methylphenoxy)-

impurity 2

Reference substance name:

1-{[2-hydroxy-3-(2-methylphenoxy)propyl][3-({[2-hydroxy-3-(2-methylphenoxy)propyl]amino}methyl)benzyl]amino}-3-(2-methylphenoxy)propan-1-ol

Molecular formula:

C38H48N2O6

IUPAC Name:

1-{[2-hydroxy-3-(2-methylphenoxy)propyl][3-({[2-hydroxy-3-(2-methylphenoxy)propyl]amino}methyl)benzyl]amino}-3-(2-methylphenoxy)propan-1-ol

impurity 3

Reference substance name:

3,3',3'',3'''-[benzene-1,3-diylbis(methanediylnitrilo)]tetrakis[1-(2-methylphenoxy)propan-2-ol]

Molecular formula:

C48H60N2O8

IUPAC Name:

3,3',3'',3'''-[benzene-1,3-diylbis(methanediylnitrilo)]tetrakis[1-(2-methylphenoxy)propan-2-ol]

Test material form:

liquid: viscous

Details on test material:

Identification:
1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Appearance/Physical state:
clear, colorless, viscous liquid
Batch:
WA 1508
Purity:
100% UVCB
Expiry date:
01 January 2021
Storage conditions:
room temperature, in the dark

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: 1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Batch: WA 1508
Purity: Not provided
Physical state/Appearance: Clear colorless viscous liquid
Expiry Date: 01 January 2021
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

Range-Finding Tests
A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Test solutions

Vehicle:

no

Details on test solutions:

Range-Finding Tests
The loading rates to be used in the definitive test were determined by preliminary range-finding tests.
The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 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 were prepared for each control and test concentration.
Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.0 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding tests a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 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.
The results of the initial range-finding test showed significant inhibition of growth occurred at both 10 and 100 mg/L loading rate WAF and so a second range finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.010, 0.10 and 1.0 mg/L for a period of 72 hours. Due to the need to test at relatively low test concentrations, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give further test concentrations of 0.10 and 0.010 mg/L.
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give a 1.0 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.10 and 0.010 mg/L loading rate WAF.
An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.6 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF.
Exposure conditions in the second range-finding test were the same as those in the initial test.
A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive Test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L.
Experimental Preparation
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for 1-Hour. Visual observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Filtration through two sheets of filter paper was also required to ensure all undissolved test item was removed. Microscopic observations of the WAF were performed after filtering and showed there to be no micro-dispersions of test item present.
A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.50, 0.25, 0.125 and 0.0625 mg/L loading rate WAF. An aliquot (1 liter) of each of the loading rate WAFs was separately inoculated with algal suspension (6.4 mL) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L loading rate WAF.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (below). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2”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 (100 to 150 rpm) and constant illumination at 24 ± 1”C until the algal cell density was approximately 10^4 to 10^5 cells/mL

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

not reported

Test temperature:

Temperature was maintained at 24 ± 1 °C

pH:

The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 8.0 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 reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.023 mg/L, to 0.16 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 0.083 mg/L.
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Details on test conditions:

Experimental Design and Study Conduct
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

Range-Finding Tests
The loading rates to be used in the definitive test were determined by preliminary range-finding tests.
The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 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 were prepared for each control and test concentration.
Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.0 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding tests a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 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.
The results of the initial range-finding test showed significant inhibition of growth occurred at both 10 and 100 mg/L loading rate WAF and so a second range finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.010, 0.10 and 1.0 mg/L for a period of 72 hours. Due to the need to test at relatively low test concentrations, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give further test concentrations of 0.10 and 0.010 mg/L.
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give a 1.0 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.10 and 0.010 mg/L loading rate WAF.
An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.6 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF.
Exposure conditions in the second range-finding test were the same as those in the initial test.
A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L.

Experimental Preparation
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for 1-Hour. Visual observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Filtration through two sheets of filter paper was also required to ensure all undissolved test item was removed. Microscopic observations of the WAF were performed after filtering and showed there to be no micro-dispersions of test item present.
A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.50, 0.25, 0.125 and 0.0625 mg/L loading rate WAF. An aliquot (1 liter) of each of the loading rate WAFs was separately inoculated with algal suspension (6.4 mL) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours (see Annex 4).

Exposure Conditions
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.86 x 105 cells per mL. Inoculation of 1 liter of test medium with 6.4 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Assessments
Test Organism Observations
Samples were taken at 20, 48 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 item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria
The pH of the control and each test concentration loading rate WAF 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.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.062 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Yield

Details on results:

Validation of Mixing Period
Preliminary investigational work indicated that there was a significant increase in the amount of dissolved test item obtained when the preparation period was extended from 24 to 96 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period

Range-finding Tests
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range finding tests are given in Table 1 and Table 2.
The results showed no effect on growth at 0.010 and 0.10 mg/L loading rate WAF. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L loading rate WAF.
Based on this information loading rates of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L were selected for the definitive test.
Chemical analysis of the 0.10 and 1.0 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations of less than the LOQ, determined to be 0.023 mg/L, and 0.26 mg/L respectively were obtained. Measured concentrations of less than the LOQ and 0.24 mg/L were obtained at 72 hours.

Definitive Test
Chemical Analysis of Test Loading Rates
Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.023 mg/L, to 0.16 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 0.083 mg/L.
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Growth Data
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 3.
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72 Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErL10 (0 - 72 h) : 0.19 mg/L loading rate WAF
ErL20 (0 - 72 h) : 0.36 mg/L loading rate WAF
ErL50 (0 - 72 h) : 1.1 mg/L loading rate WAF

Where ErLx is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all loading rates 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, 0.0625 and 0.125 mg/L loading rate WAFs (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 0.125 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 0.25 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h) : 0.053 mg/L loading rate WAF
EyL20 (0 - 72 h) : 0.091 mg/L loading rate WAF
EyL50 (0 - 72 h) : 0.23 mg/L loading rate WAF; 95% confidence limits 0.19 to 0.27 mg/L loading rate WAF

Where EyLx is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out as above. There were no statistically significant differences between the control and 0.0625 mg/L loading rate WAF (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on yield was 0.0625 mg/L loading rate WAF. Correspondingly the LOEL based on yield was 0.125 mg/L loading rate WAF.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 271 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 : 1.08 x 10^6 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 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

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

Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 8.0 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of stirring the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless media column with test item adhered to the glass slide suspended within the media column. After stirring the WAF was observed to have formed a clear colorless media column with test item adhered to the glass slide suspended within the media column, some white flakes were observed dispersed throughout the media column. After standing for 1-Hour the WAF was observed to be as at the end of stirring, however, most of the white flakes were observed to have settled on the bottom of the mixing vessel. After filtration through a glass wool plug and two sheets of filter paper, no micro-dispersions of test item were observed.
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, 0.0625, 0.125 and 0.25 mg/L loading rate WAFs were observed to be green dispersions. The 0.50 mg/L loading rate WAF test cultures were observed to be very pale green dispersions whilst the 1.0 mg/L loading rate WAF test cultures were observed to be extremely pale green dispersions.

Results with reference substance (positive control):

A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

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

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 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.

Any other information on results incl. tables

Table 2            Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/L)		pH	Cell Densities*(cells per mL)			pH
		0 h	20 h	48 h	72 h	72 h
Control	R1	7.3	3.17E+04	2.55E+05	1.30E+06	8.0
	R2		3.43E+04	2.52E+05	1.33E+06
	R3		3.09E+04	2.47E+05	1.34E+06
	R4		3.26E+04	2.73E+05	1.47E+06
	R5		3.36E+04	2.62E+05	1.29E+06
	R6		3.29E+04	2.44E+05	1.41E+06
	Mean		3.27E+04	2.55E+05	1.36E+06
0.0625	R1	7.4	3.07E+04	2.24E+05	9.62E+05	8.1
	R2		3.03E+04	2.15E+05	1.26E+06
	R3		3.05E+04	2.05E+05	1.23E+06
	Mean		3.05E+04	2.15E+05	1.15E+06
0.125	R1	7.4	2.58E+04	1.67E+05	9.97E+05	8.1
	R2		2.42E+04	1.81E+05	9.33E+05
	R3		2.88E+04	1.76E+05	9.55E+05
	Mean		2.63E+04	1.75E+05	9.61E+05
0.25	R1	7.3	2.13E+04	1.10E+05	5.59E+05	8.0
	R2		2.63E+04	1.63E+05	8.77E+05
	R3		1.96E+04	1.08E+05	5.53E+05
	Mean		2.24E+04	1.27E+05	6.63E+05
0.50	R1	7.3	1.79E+04	8.20E+04	3.18E+05	7.9
	R2		1.83E+04	5.98E+04	2.44E+05
	R3		1.78E+04	7.22E+04	2.96E+05
	Mean		1.80E+04	7.13E+04	2.86E+05
1.0	R1	7.2	1.28E+04	7.20E+04	1.50E+05	7.8
	R2		1.45E+04	3.33E+04	9.63E+04
	R3		1.37E+04	3.07E+04	7.35E+04
	Mean		1.37E+04	4.54E+04	1.07E+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: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 hour	% Inhibition	0 to 72 hour	% Inhibition*
Control	R1	0.077		1.29E+06
	R2	0.078		1.32E+06
	R3	0.078		1.34E+06
	R4	0.079	-	1.46E+06	-
	R5	0.077		1.28E+06
	R6	0.078		1.41E+06
	Mean	0.078		1.35E+06
	SD	0.001		7.05E+04
0.0625	R1	0.073	6	9.57E+05
	R2	0.077	1	1.26E+06
	R3	0.076	3	1.22E+06
	Mean	0.075	3	1.15E+06	15
	SD	0.002		1.65E+05
0.125	R1	0.074	5	9.92E+05
	R2	0.073	6	9.28E+05
	R3	0.073	6	9.50E+05
	Mean	0.073	6	9.56E+05	29
	SD	0.001		3.24E+04
0.25	R1	0.066	15	5.54E+05
	R2	0.072	8	8.72E+05
	R3	0.065	17	5.48E+05
	Mean	0.068	13	6.58E+05	51
	SD	0.004		1.85E+05
0.50	R1	0.058	26	3.13E+05
	R2	0.054	31	2.39E+05
	R3	0.057	27	2.91E+05
	Mean	0.056	28	2.81E+05	79
	SD	0.002		3.82E+04
1.0	R1	0.047	40	1.45E+05
	R2	0.041	47	9.13E+04
	R3	0.037	53	6.85E+04
	Mean	0.042	47	1.02E+05	92
	SD	0.005		3.95E+04

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

R=   Replicate

SD= Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The data satisfied the validation criterion given in the OECD Guideline

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:
Response Variable EL50(mg/L Loading Rate WAF) 95% Confidence Limits (mg/L Loading Rate WAF) No Observed Effect Loading Rate (NOEL) (mg/L) Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate 1.1 * 0.125 0.25
Yield 0.23 0.19 - 0.27 0.0625 0.125

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. 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) 761/2009.

Methods…

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following preliminary range-finding tests,Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates 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 less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.023 mg/L, to 0.16 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 0.083 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure ofPseudokirchneriella subcapitatato the test item gave the following results:

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)			No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	1.1		*		0.125	0.25
Yield	0.23	0.19	-	0.27	0.0625	0.125

*It was not possible to calculate 95% confidence limits for the E_rL₅₀value as the data generated did not fit the models available for the calculation of confidence limits