Reaction products of fatty acids, C18 (unsaturated) alkyl and epoxidized fatty esters, with amines polyethylenepoly-, tetraethylenepentamine

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 start date 16 February 2021 and Experimental completion date 13 July 2021

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)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis.
An additional preparation of each test concentration was incubated alongside the test from which samples were removed for analysis at 24 and 48 hours.
A set of duplicate samples were taken on each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): 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 was 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 105 to 106 cells/mL.

Test type:

static

Water media type:

freshwater

Remarks:

The culture medium used for both the range-finding tests, initial experiment and definitive test was the same as that used to maintain the stock culture.

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

no

Post exposure observation period:

no

Test temperature:

24°C

pH:

The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Dissolved oxygen:

n/a

Salinity:

not applicable

Conductivity:

n/a

Nominal and measured concentrations:

Nominal concentration in the Definitive Test: 0.00625 / 0.0125 / 0.025 / 0.050 / 0.10 mg/L
Geometric Mean Measured test concentration in the Definitive tests: 0 / 0.0013 / 0.0017 / 0.0019 / 0.0021 / 0.0045 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed (plugged with polyurethanne foam bungs to reduce evaporation)
- Material, size, headspace, fill volume: 100 mL of test preparation
- The test item was dissolved directly in culture medium
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 5.0 x 10E3 celles per mL.
- No. of vessels per concentration (replicates): 3 replicate flasks each containing 100 mL were used for each treatment group.
- No. of vessels per control (replicates): 6 replicates containing 100 mL
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination provided by warm white lughting (380 to 730 nm) and constantly shaken at apprx. 150 rpm for 72 hours
- Light intensity and quality: Appr. 7000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities was determined using a Coulter Multisizer Particle Counter. Three determinations were made for each sample. The inoculated cell concentration (5.00x10E3 cells /ml was taken as the starting cell density
- Other: Shape size of the algal cells was inspected microscopically and any abnormalities recorded.
- The pH of the control and each 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.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- For the first range-finding test: spacing factor is 10
- For the second range-finding test: spacing factor is 10
- For the Definitive Test: spacing factor is 2

- Range finding study: 2 range finding test were done

- Test concentrations:
Nominal concentration first range-finding test: 0 / 0.10 / 1.0 / 10 / 100 mg/L

Nominal concentration second range-finding test: 0 / 0.0010 / 0.010 / 0.10 / 1.0 mg/l
- Results used to determine the conditions for the definitive study:
Chemical analysis of the test preparations taken from the second range-finding test at 0 hours showed measured test concentrations to range from less than the LOD at 0.0010 mg/L, to 101% of nominal at 1.0 mg/L.
Analysis of the corresponding preparations at 72 hours showed measured test concentrations to range from less than the LOD at 0.0010 mg/L to 79% of nominal at 1.0 mg/L.
Given the decline in measured test concentrations observed over the 72-Hour test period it was considered justifiable to provide samples for analysis from the definitive test at 0, 24, 48 and 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits: 0.00042 - 0.0014 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits: 0.00082 - 0.0018 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.003 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits: 0.0022 - 0.0040 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Tests:
The results showed no significant effect on growth at the test concentrations of 0.0010 and 0.010 mg/L. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100 mg/L.
Based on this information test concentrations of 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L were selected for the definitive test.


Definitive Test.
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of detection (LOD) of the analytical method employed, determined to be 0.0025 mg/L, to 0.037 mg/L. A decline in measured test concentrations was observed to less than the LOD in all test preparations at 24 and 48 hours, and from less than the LOD to 0.0074 mg/L at 72 hours. These results indicate that the test item was unstable under the conditions of the test.

Inspection of the data could find no apparent cause for not having achieved near nominal test concentrations in the test preparations taken for analysis at 0 hours. Given that this effect had also been observed from analysis of the test preparations taken from the initial experiment, and that at the measured concentrations obtained, significant inhibition of growth occurred to enable the calculation of the required test endpoints, this was considered to have had no adverse effect on the outcome or integrity of the test.

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. In cases where the measured concentration was less than the LOD of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.00125 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations were determined to be:


Nominal Test Concentration in mg/l Geometric Mean Measured Test Concentration (mg/L)
0.00625 0.0013
0.0125 0.0017
0.025 0.0019
0.050 0.0021
0.10 0.0045

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using Williams Multiple Sequential t-test Procedure. There were no statistically significant differences (P0.05), between the control and 0.0013 mg/L test concentration; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.0013 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 0.0017 mg/L.

Results with reference substance (positive control):

A positive control (Covance study number 8461000) used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L.
The positive control was conducted between 21 December 2020 and 24 December 2020.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.5 mg/L
EyC50 (0 to 72 hour) : 0.44 mg/L; 95% confidence limits 0.37 to 0.52 mg/L

No Observed Effect Concentration based on growth rate: 0.125 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.25 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item*.

Reported statistics and error estimates:

The following computerized systems were used in the study:
Coulter® Multisizer 3 Particle Counter (3.53) Cell density determinations
Delta control system (Version 3.4.0) Building management
ToxRat (Version 3.3.0) Professional computer software package
Veeva QMS Electronic communication system

        

The results of the test are considered valid if the following performance criteria 

The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (Days 0 to 1, 1 to 2 and 2 to 3, for the 72 -hours tests) must not exceed 35%.

The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%

 

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 174 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 10³cells per mL

Mean cell density of control at 72 hours                  :          8.70 x 10⁵cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 6% 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 hour) was 3% 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 0.0013, 0.0017 and 0.0019 mg/L. Slightly enlarged cells were observed to be present in the 0.0021 mg/L test cultures whilst in the 0.0045 mg/L test cultures enlarged and damaged cells were observed.

Water Quality Criteria

The pH values of the control and each test preparation are given inTable 3. 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 8.4 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 the control cultures were observed to be green dispersions. The 0.0013 and 0.0017 mg/L test cultures were slightly paler green dispersions when compared to the control. The 0.0019 mg/L test cultures were very pale green dispersions, the 0.0021 mg/L test cultures were extremely pale green dispersions whilst the 0.0045 mg/L test cultures were either clear colourless solutions or very pale green dispersions. 

Validity criteria fulfilled:

yes

Conclusions:

ErC50 and NOEC of the test item on the growth of the green alga Raphidocelis subcapitata was 0.0027 mg/l and 0.0013 mg/l respectively.

Executive summary:

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 followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.

Following preliminary range-finding tests and an initial experiment, Raphidocelis subcapitata was exposed to aqueous solutions of the test item at nominal concentrations of 0.00625, 0.0125, 0.025, 0.050 and 0.10 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of detection (LOD) of the analytical method employed, determined to be 0.0025 mg/L, to 0.037 mg/L. A decline in measured test concentrations was observed to less than the LOD in all test preparations at 24 and 48 hours, and from less than the LOD to 0.0074 mg/L at 72 hours. These results indicate that the test item was unstable under the conditions of the test.

Given this decline in measured test concentrations it was 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 0.0013, 0.0017, 0.0019, 0.0021 and 0.0045 mg/L.

Exposure of Raphidocelis subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	Endpoint	Measured Concentration (mg/L)	95% Confidence limits (mg/L)
Growth Rate	ErC10	0.0011	0.00042 – 0.0014
	ErC20	0.0014	0.00082 – 0.0018
	ErC50	0.0027	0.0022 – 0.0040
	NOEC	0.0013	Not applicable
	LOEC	0.0017	Not applicable

 

Response Variable	Endpoint	Measured Concentration (mg/L)	95% Confidence limits (mg/L)
Yield	EyC10	0.0011	0.00015 – 0.0014
	EyC20	0.0012	0.00029 – 0.0015
	EyC50	0.0015	0.00097 – 0.0018
	EyC90	0.0022	0.0018 – 0.0081
	NOEC	<0.0013	Not applicable
	LOEC	≤0.0013	Not applicable

Description of key information

ErC50 and NOEC of the test item on the growth of the green alga Raphidocelis subcapitata was 0.0027  mg/l and 0.0013 mg/l respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.004 mg/L

EC10 or NOEC for freshwater algae:

0.001 mg/L