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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 12 June 2017. Experimental completion date 05 July 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Identification: 1-(methylamino)-4-[(3-methylphenyl)amino] anthraquinone
Physical state/Appearance: dark red powder
Batch: 702W01
Purity: 99.4%
Expiry Date: 08 February 2020
Storage Conditions: room temperature in the dark
Intended use/Application: industrial chemical
Specific details on test material used for the study:
Identification: 1-(methylamino)-4-[(3-methylphenyl)amino] anthraquinone
Physical state/Appearance: Dark red powder
Batch: 702W01
Purity: 99.4%
Expiry Date: 08 February 2020
Storage Conditions: 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
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.

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 (see Annex 3).

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
It was evident from these results that a decline in measured concentrations occurred when the preparation period was extended beyond 24 hours. Whilst the centrifuged test samples yielded the highest dissolved test item concentrations, visual inspection of the 48Hour sample centrifuged at 40000 g showed undissolved test item remained and as such it is considered that centrifugation was not sufficient to ensure all 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 filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a 100% v/v saturated solution of the test item.

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 filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) 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 (4.6 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 filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. An aliquot (1 liter) of the stock solution was inoculated with 21.4 mL of algal suspension to give the required test concentration of 100% v/v saturated solution.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

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 (Section 3.3). 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 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 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:
7.8 - 8.0
Nominal and measured concentrations:
Range-Finding Test
nominal test concentrations of 1.0, 10 and 100% v/v saturated solution

Definitive Test
100% v/v saturated solution

Test concentrations of less than the limit of quantification (LOQ)
Details on test conditions:
Range-Finding Test
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 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.

Definitive Test
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 2.34 x 105 cells per mL. Inoculation of 1 liter of test medium with 21.4 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 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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution.
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v saturated solution.
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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.
Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 72 hours (see Annex 4) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.057 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours (see Annex 4) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.057 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.
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 nominal test concentration of 100% v/v saturated solution over the 72-Hour exposure period
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h): >100% v/v saturated solution
ErC20 (0 - 72 h): >100% v/v saturated solution
ErC50 (0 - 72 h): >100% v/v saturated solution
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 100% v/v saturated solution 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 differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution.

Inhibition of Yield
EyC10 (0 - 72 h): >100% v/v saturated solution
EyC20 (0 - 72 h): >100% v/v saturated solution
EyC50 (0 - 72 h): >100% v/v saturated solution
Where:
EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as above. There were no statistically significant differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution.
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.

Any other information on results incl. tables

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 201 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 : 1.00 x 106 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 7.8 at 0 hours to pH 7.9 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

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 and 100% v/v saturated solution test cultures were observed to be green dispersions

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 gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.
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 saturated solution method of preparation was appropriate for this test item.

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 filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) 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 test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.057 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated soltuion.

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