Reaction products of diethylenetriamine and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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 01 August 2017 and 09 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:

EDA-BADGE-EDA

Cas Number:

854009-15-7

Molecular formula:

C25H40N4O4

IUPAC Name:

EDA-BADGE-EDA

Constituent 2

Reference substance name:

EDA-BADGE-EDA-BADGE-EDA

Molecular formula:

C48H72N6O8

IUPAC Name:

EDA-BADGE-EDA-BADGE-EDA

Constituent 3

Reference substance name:

EDA-BADGE α-glycol

Molecular formula:

C23H34N2O5

IUPAC Name:

EDA-BADGE α-glycol

Constituent 4

Reference substance name:

reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

IUPAC Name:

reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

Constituent 5

Reference substance name:

Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

IUPAC Name:

Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine
Physical state/Appearance: Yellow/brown extremely viscous liquid
Batch: BBF01102V1
Purity: Not provided
Expiry Date: 01 January 2021
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Definitive Test
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 Test
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. Prior to use the test item was heated to 80 ºC in order to aid handling.
Nominal amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 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 23 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 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 75-100 mL discarded) to give the 10 and 100 mg/L loading rate. Microscopic observations of the WAFs were performed after filtering twice and showed no micro-dispersions of test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (8.4 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.

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 the remaining test concentrations. Prior to use the test item was heated to 80 ºC in order to aid handling.
A nominal amount of test item (20 mg) was weighed onto a glass slide and suspended within 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 23 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 75-100 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 (6.6 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF

Definitive Test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Prior to use the test item was heated to 80 ºC in order to aid handling.
Experimental Preparation
A nominal amount of test item (20 mg) was weighed onto a glass slide and suspended within 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 23 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 75-100 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 0.32, 0.10, 0.032 and 0.010 mg/L loading rate WAF.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.2 mL) to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF.
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours

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 (Section 3.3). The master cultures were kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 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 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:

yes

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 7.4 at 0 hours to pH 8.2 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.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Total Organic Carbon Analysis
Total Organic Carbon (TOC) analysis of the test preparations at 0 hours (see Annex 4) showed measured carbon concentrations to range from less than the limit of quantification (LOQ), determined to be 1.0 mg C/L to 9.6 mg C/L. The measured concentrations obtained did not however follow a loading rate dependent pattern; the highest carbon concentration obtained was from the 0.010 mg/L loading rate WAF. Such a value was considered to be due to possible contamination of the test vessel within which the sample was placed. Due to an instrument malfunction, no results were available from the 72-Hour test preparations. This was considered to have had no impact on the outcome of the test given that TOC analysis is not stability indicating and that all results were to be 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 levels of total organic carbon, as an indicator of soluble organic substances 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. Prior to use the test item was heated to 80 ºC in order to aid handling.
Nominal amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 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 23 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 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 75-100 mL discarded) to give the 10 and 100 mg/L loading rate. Microscopic observations of the WAFs were performed after filtering twice and showed no micro-dispersions of test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (8.4 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 test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
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 the remaining test concentrations. Prior to use the test item was heated to 80 ºC in order to aid handling.
A nominal amount of test item (20 mg) was weighed onto a glass slide and suspended within 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 23 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 75-100 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 (6.6 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 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 test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a haemocytometer and light microscope.

Definitive Test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Prior to use the test item was heated to 80 ºC in order to aid handling.

Experimental Preparation
A nominal amount of test item (20 mg) was weighed onto a glass slide and suspended within 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 23 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 75-100 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 0.32, 0.10, 0.032 and 0.010 mg/L loading rate WAF.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.2 mL) to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF.
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours.

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 500 mL of test medium with 3.2 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Assessments
Test Organism Observations
Samples were taken at 22, 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 10^3 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 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.:

> 0.31 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.:

0.1 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.:

0.32 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.:

0.16 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.:

0.1 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.:

0.32 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 total organic carbon when the preparation period was extended from 24 to 96 hours. However, visual observations made on the 96-Hour test preparation after filtration showed that dispersed test item remained. The presence of this dispersed material may have exerted a physiological effect on the test organisms. Given this, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range-finding Test
The results showed no effect on growth at 0.010 mg/L loading rate WAF. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100 mg/L loading rate WAF.
Based on this information loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were selected for the definitive test.

Definitive Test
Total Organic Carbon Analysis
Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed measured carbon concentrations to range from less than the limit of quantification (LOQ), determined to be 1.0 mg C/L to 9.6 mg C/L. The measured concentrations obtained did not however follow a loading rate dependent pattern; the highest carbon concentration obtained was from the 0.010 mg/L loading rate WAF. Such a value was considered to be due to possible contamination of the test vessel within which the sample was placed. Due to an instrument malfunction, no results were available from the 72-Hour test preparations. This was considered to have had no impact on the outcome of the test given that TOC analysis is not stability indicating and that all results were to be based on nominal loading rates only.

Growth Data
Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 1.
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) : 0.26 mg/L loading rate WAF
ErL20 (0 - 72 h) : 0.28 mg/L loading rate WAF
ErL50 (0 - 72 h) : 0.31 mg/L loading rate WAF; 95% confidence limits 0.30 – 0.32 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, 0.010, 0.032, 0.10 and 0.32 mg/L loading rate WAF test groups 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, 0.010, 0.032 and 0.10 mg/L loading rate WAFs (P>0.05), however the 0.32 mg/L loading rate was significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 0.10 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 0.32 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h) : 0.11 mg/L loading rate WAF
EyL20 (0 - 72 h) : 0.12 mg/L loading rate WAF
EyL50 (0 - 72 h) : 0.16 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 decreases in yield between the control, 0.010, 0.032 and 0.10 mg/L loading rate WAFs (P>0.05), however the 0.32 mg/L loading rate was significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 0.10 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 0.32 mg/L loading rate WAF.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 216 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 103 cells per mL
Mean cell density of control at 72 hours : 1.08 x 106 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 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. After 72 hours there were no abnormalities detected in the control or test cultures at 0.010, 0.032 and 0.10 mg/L loading rate WAF, however some misshapen cells were observed to be present in the test cultures at 0.32 mg/L loading rate WAF and cell fragments and only a few intact cells were observed to be present in the test cultures at 1.0 mg/L loading rate WAF.

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 7.4 at 0 hours to pH 8.2 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.

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 the mixing period the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless media column with test item visible adhered to the glass slide. After stirring a clear colorless media column with solidified test item dispersed throughout and floating at the media surface was formed. Following a 1-Hour standing period the WAF had formed a clear colorless media column with solidified test item floating at the media surface and settled on the bottom of the mixing vessel. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present.
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, 0.010, 0.032 and 0.10 mg/L loading rate WAF test cultures were observed to be green dispersions whilst the 0.32 and 1.0 mg/L loading rate WAF test cultures were observed to be clear colorless solutions

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 1: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control[ ]	R1	0.074		1.05E+06
	R2	0.074		1.05E+06
	R3	0.073		9.61E+05
	R4	0.076	-	1.21E+06	-
	R5	0.075		1.10E+06
	R6	0.074		1.06E+06
	Mean	0.074		1.07E+06
	SD	0.001		8.12E+04
0.010	R1	0.078	[5]	1.41E+06
	R2	0.077	[4]	1.25E+06
	R3	0.078	[5]	1.37E+06
	Mean	0.078	[5]	1.35E+06	[25]
	SD	0.001		8.27E+04
0.032	R1	0.075	[1]	1.08E+06
	R2	0.080	[8]	1.57E+06
	R3	0.078	[5]	1.34E+06
	Mean	0.078	[5]	1.33E+06	[24]
	SD	0.003		2.45E+05
0.10	R1	0.075	[1]	1.09E+06
	R2	0.074	0	1.06E+06
	R3	0.075	[1]	1.12E+06
	Mean	0.075	[1]	1.09E+06	[2]
	SD	0.001		3.31E+04
0.32	R1	0.041	45	9.09E+04
	R2	0.026	65	2.82E+04
	R3	0.022	70	1.93E+04
	Mean	0.030	60	4.61E+04	96
	SD	0.010		3.90E+04
1.0	R1	-0.005	107	-1.48E+03
	R2	-0.006	108	-1.74E+03
	R3	-0.007	109	-1.88E+03
	Mean	-0.006	108	-1.70E+03	100
	SD	0.001		2.03E+02

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

[Increase in growth as compared to controls]

R₁-R₆= Replicates 1 to 6

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 0.31 0.30 - 0.32 0.10 0.32
Yield 0.16 * 0.10 0.32

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 0.010, 0.032, 0.10, 0.32 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

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed measured carbon concentrations to range from less than the limit of quantification (LOQ), determined to be 1.0 mg C/L to 9.6 mg C/L. The measured concentrations obtained did not however follow a loading rate dependent pattern; the highest carbon concentration obtained was from the 0.010 mg/L loading rate WAF. Such a value was considered to be due to possible contamination of the test vessel within which the sample was placed. Due to an instrument malfunction, no results were available from the 72-Hour test preparations. This was considered to have had no impact on the outcome of the test given that TOC analysis is not stability indicating and that all results were to be 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	0.31	0.30	-	0.32	0.10	0.32
Yield	0.16		*		0.10	0.32

*It was not possible to calculate 95% confidence limits for the E_yL₅₀value as the data generated did not fit the models available for the calculation of confidence limits.