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Ecotoxicological information

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:
Experimental start date 06 February 2017 Experimental completion date 11 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2H-benzotriazol-2-yl)-4-tert-butylphenol
EC Number:
221-574-0
EC Name:
2-(2H-benzotriazol-2-yl)-4-tert-butylphenol
Cas Number:
3147-76-0
Molecular formula:
C16H17N3O
IUPAC Name:
2-(5-tert-Butyl-2-hydroxyphenyl)benzotriazole
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 02002IX3
- Expiration date of the lot/batch: 25 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range-Finding Test
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours
Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Two additional samples of the 100% v/v saturated solution test group were prepared at the start of the test and incubated alongside the test to provide samples for analysis at 24 and 48 hours.

Test solutions

Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial
Information provided by the Sponsor indicated the test item was insoluble in water.
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions

Saturated Solution Preparation
A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)

Discussion
The results from the filtered test samples suggest that the test item was possibly adsorbing to the filter matrices. There was no significant increase in the dissolved test item concentration obtained from the centrifuged test samples when the preparation period was extended beyond 24 hours. Centrifugation at 40000 g was considered most appropriate as the higher measured concentration obtained from the 24-Hour 10000 g sample indicated that not all of the undissolved test item was removed.
Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by centrifugation at 40000 g for 30 minutes to give a nominal test concentration of approximately 0.028 mg/L.

Range-Finding Test
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10 and 1.0% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.5 mL) to give the required test concentrations of 1.0, 10 and 100% v/v saturated solution.

Definitive Test
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution.
An aliquot (1 liter) of this saturated solution was inoculated with algal suspension (14.4 mL) to give the required test concentration of 100% v/v saturated solution.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 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 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test.
pH:
The pH of the test solution was 8.0 - 8.4 at 0 and 72 hours respectively.
The pH of the control and 100% v/v saturated solution test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.1 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.
Nominal and measured concentrations:
Range finding test
Nominal: 1.0, 10 and 100% v/v saturated solution.
Chemical analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 0.10 mg/L. A decline in measured concentration was observed at 72 hours to 0.029 mg/L indicating that the test item was unstable over the test period.

Defintive test
Nominal: 100% v/v saturated solution test
Chemical analysis of the 100% v/v saturated solution test preparations at 0, 24, 48 and 72 hours (see Annex 5) showed measured test concentrations of 0.58, 0.34, 0.35 and 0.19 mg/L respectively were obtained indicating that the test item was unstable over the test duration.
Details on test conditions:
Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.028 mg/L could be obtained using a saturated solution method of preparation.
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution 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 used for each control and test concentration.
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.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. 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 test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration no effect on algal growth was observed.
Experimental Preparation
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and 100% v/v saturated solution 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 3.48 x 10^5 cells per mL. Inoculation of 1 liter of test medium with 14.4 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 23, 46 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 100% v/v saturated solution test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.

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.
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.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Range-finding Test
The results showed no effect on growth at the test concentrations of 1.0, 10 and 100% v/v saturated solution.
Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.
The measured concentration obtained from the 100% v/v saturated solution at 0 hours (0.10 mg/L) was significantly higher than that obtained from the preliminary media preparation trial (0.028 mg/L). This was considered to be due mainly to a test media effect whereby the test item was more readily soluble in culture medium than in deionized reverse osmosis water. Furthermore, due to the need to use centrifugation as a means to remove any dispersed test item present, some variability in the obtained concentration was considered likely. This was considered to have had no adverse effect on the outcome of the test as no inhibitory effects were observed at the higher attained concentration.

Definitive Test
Verification of Test Concentrations
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. The geometric mean measured test concentration was determined to be 0.34 mg/L.

Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 0.34 mg/L over the 72-Hour exposure period.
The test concentration of 0.34 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility and stability of the test item in water.
Accordingly the following results were determined from the data based on the geometric man measured test concentration:

Inhibition of Growth Rate
ErC10 (0 - 72 h): >0.34 mg/L
ErC20 (0 - 72 h): >0.34 mg/L
ErC50 (0 - 72 h): >0.34 mg/L
Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 0.34 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant decreases in growth rate (P≥0.05), between the control and 0.34 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.34 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): >0.34 mg/L
EyC20 (0 - 72 h): >0.34 mg/L
EyC50 (0 - 72 h): >0.34 mg/L
Where:
EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out. There were no statistically significant decreases in yield (P≥0.05), between the control and 0.34 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.34 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 93 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 : 4.66 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 11% 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 4% 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.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period control replicate R1 was observed to be a pale green dispersion whilst replicates R2-R6 were observed to be green dispersions. The test cultures were observed to be bright green dispersions.
Results with reference substance (positive control):
A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

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

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

ErC50 (0 – 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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and, based on the geometric mean measured test concentration gave EC50 values of greater than 0.34 mg/L. The No Observed Effect Concentration was 0.34 mg/L.
This study showed that there were no toxic effects at saturation.
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. 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 Regulation (EC) No 761/2009.

Methods

Information provided by the Sponsor indicated the test item was insoluble in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.028 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solution was prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to produce a 100% v/v saturated solution of the test item.

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 100% v/v saturated solution test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of 0.58, 0.34, 0.35 and 0.19 mg/L respectively were obtained indicating that the test item was unstable over the test duration.

Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.34 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 0.34 mg/L. The No Observed Effect Concentration was determined to be 0.34 mg/L.

This study showed that there were no toxic effects at saturation.