N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 21 July 2020 to 14 September 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations

Samples were taken from the control and each test group from the bulk test preparation at 0 hours, from additional media run alongside the test at 24 and 48 hours, and from the pooled replicates at 72 hours for quantitative analysis. All samples were sent for analysis on the day of sampling. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary. All sample bottles were fortified by the addition of 10 mL of acetonitrile in order to stabilise the test item.

Vehicle:

no

Remarks:

Saturate solution used

Details on test solutions:

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 categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019) and a preliminary media preparation trial was conducted as follows:

A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 litres of ASTM media 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 Gelman Acrocap filter (approximately 100 mL used to pre-condition the filter was discarded)
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 500 mL used to pre-condition the filter was discarded)


Based on the information obtained as part of the preliminary media preparation trial for the main study 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 Gelman Acrocap filter (first approximate 100 mL discarded) to give a nominal test concentration of approximately 0.22 mg/L.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at 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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ±1 ºC

pH:

pH ranged from 7.6 at 0 hours to 8.6 at 72-hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Material, size, headspace, fill volume: Each conical flasks contained 100 ml of solution.
- Aeration: The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

- Initial cells density: initial nominal cell density of 5.00 x 1063 cells per mL
- No. of vessels per concentration (replicates): three flasks were used for each treatment group
- No. of vessels per control (replicates): Six flasks were used for the control group and
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: Yes
- Photoperiod: Constant illumination at 24 ±1°C
- Light intensity and quality: warm white lighting (380 to 730 nm) at approximately 7000 lux

Reference substance (positive control):

yes

Remarks:

Potassium dichromate run as a separate study (20 January 2020 to 06 March 2020) to confirm test system was responding as expected. The results from the positive control study were within the normal ranges for this reference item.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

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 0.0014, 0.015, 0.041 and 0.12 mg/L, however enlarged cells were observed to be present in the test cultures at 0.0051 mg/L.

Results with reference substance (positive control):

A positive control using potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L was run as a separate study between 20 January 2020 and 06 March 2020. The results from the positive control with potassium dichromate were within the normal ranges for this reference item in the laboratory.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using a Shapiro-Wilk’s Test on Normal Distribution followed by Levene’s Test on Variance Homogeneity, Trend analysis by Contrasts and Dunnett’s Multiple t-test Procedure.

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 test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.

 

Chemical analysis of the test preparations showed measured test concentrations of 0.0012 to 0.12 mg/L at 0 hours. Measured test concentrations of less than the limit of detection (LOD) of the analytical method employed, which was determined to be 0.0001 mg/L, to 0.00066 were obtained at 72 hours, indicating that the test item was not stable under test conditions.

 

Definitive Test

 

Verification of Test Concentrations

 

Due to failure of the original procedural recoveries at 0 hours, the frozen duplicates were thawed and analysed with a fresh calibration standard and diluent, and the duplicate values used for reporting purposes.

 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0018 to 0.17 mg/L. Measured test concentration ranging from 0.0014 to 0.14 mg/L were observed at 24 hours, 0.0015 to 0.082 mg/L at 48 hours and 0.00093 to 0.12 mg/L at 72 hours.

 

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 concentrations were determined to be:

 

 

Nominal Test Concentration (% v/v Saturated Concentration)	Geometric Mean Measured Test Concentration (mg/L)
1.0	0.0014
3.2	0.0051
10	0.015
32	0.041
100	0.12

 

 

Growth Data

From the data it is clear that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 0.12 mg/L over the 72-Hour exposure period. 

 

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 0.0014, 0.015, 0.041 and 0.12 mg/L, however enlarged cells were observed to be present in the test cultures at 0.0051 mg/L.

 

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

 

Validity Criteria
The following data show that cell concentration of the control cultures increased by a factor of 66 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 : 3.32 x 105 cells per mL

 

 The mean coefficient of variation for section by section specific growth rate for the control cultures was 28% 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 to 72 hours) was 5% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Validity criteria fulfilled:

yes

Remarks:

The test met the relevant validity criteria as required by the OECD 201 test guideline. Details of test validity are reported in the "any other information on results" section.

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated and gave EC50 values of greater than 0.12 mg/L based on the geometric mean measured test concentrations. The No Observed Effect Concentration (NOEC) was 0.12 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 Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method was designed to meet the requirements of the following guidelines:

• OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, “Growth Inhibition Test"
• Method C.3 of Commission Regulation (EC) No 761/2009 (Algal Inhibition Test)

Methods

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 0.22 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, Raphidocelis subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1°C. The test item solutions were 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 through a 0.2 µm filter to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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 0.0018 to 0.17 mg/L. Measured test concentration ranging from 0.0014 mg/L to 0.14 mg/L were observed at 24 hours, 0.0015 mg/L to 0.082 mg/L at 48 hours and 0.00093 mg/L to 0.12 mg/L at 72 hours. Therefore, 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 average geometric mean measured test concentrations for the 0, 24, 48 and 72-Hour values were determined to be 0.0014, 0.0051, 0.015, 0.041 and 0.12 mg/L.

Conclusion

Exposure of Raphidocelis subcapitata to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.12 mg/L. The No Observed Effect Concentration (NOEC) was determined to be 0.12 mg/L.

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

Description of key information

Acute toxicity to aquatic algae data summary.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.12 mg/L

EC10 or NOEC for freshwater algae:

0.12 mg/L

Additional information

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated and gave EC50 values of greater than 0.12 mg/L based on the geometric mean measured test concentrations. The No Observed Effect Concentration (NOEC) was 0.12 mg/L.

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