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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 16 February 2021 and Experimental completion date 16 June 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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 freshly prepared bulk test preparations at 0 and 24 hours and from the corresponding aged preparations (pooled replicates) at 24 and 48 hours for immediate quantitative analysis. Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium in a temperature controlled room maintaining the water temperature at
18 to 22 °C.
The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension.

Culture conditions ensured that reproduction was by parthenogenesis.

Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water (Elendt M4 Medium) used for both the range finding and definitive test.

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

not applicable

Post exposure observation period:

Not applicable

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L as CaCO3)

Test temperature:

21°C

pH:

7.9 ± 0.3

Salinity:

Alkalinity: 0.9 mmol/L

Conductivity:

not available

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L
Geometric Mean Measured Test concentration in mg/ for the definitive testl:0.042 / 0.085 / 0.14 / 0.36 / 0.80
Expression as a % of the nominal test concentration: 42 / 47 / 44 / 64 / 80

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 ml glass vessels
- Type (delete if not applicable): closed (covered to reduce evaporatio and maintained in a temperature controlled room. Maintaine between 18 to 22°C.
- Material, size, headspace, fill volume:
- Volume of solution: 100 mL
- Aeration: not aerated
- Daphnida were not individually identified and received no food during exposure.

- Renewal rate of test solution (frequency/flow rate): demi-static test condition were employed in the test in an effort to maintain dissolved test item concentrations.
Foe the test media renewal at 24 hours , the test concentrations were freshly prepared and the Daphnids transferred by wide bore pipette from the 24-hour old test media into the fresh test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable


OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the prepared media was in the range of 7.9 ± 0.3 and was stored at approximately 21 ºC. If necessary the pH was with NaOH or HCl.

- Photoperiod: 16 hours
- Light intensity: between 200 and 1200 Lux with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim within 15 secondes after gentls agitation.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits (mg/l) (0.49 - 1.2)

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

0.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range finding test:
No immobilization was observed at the test concentration of 0.10 mg/L, however; 100% immobilization was observed at 1.0, 10 and 100 mg/L.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information test concentrations of 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L were selected for the definitive test.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.081 to 94 mg/L. A concentration dependent decline in measured test concentrations was observed at 48 hours in the range of 0.0021 to 80 mg/L indicating that the test item was unstable under the conditions of the test.


Definitive test:
Analysis of the freshly prepared test solutions at 0 and 24 hours showed measured test concentrations to range from 89% to 105% of nominal.
Analysis of the corresponding aged test solutions at 24 and 48 hours showed a concentration dependent decline in measured test concentrations in the range of 18% to 76% of nominal indicating 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.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in all test concentrations. These responses were reduced mobility and being trapped at the water surface

Results with reference substance (positive control):

A positive control (Labcorp Study Number 8461001) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
The positive control was conducted between 05 January 2021 and 29 January 2021.
Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package. Accordingly the following results were obtained:

EC50 (24h)=0.86 mg/l 95% confidence limits in mg/l : 0.21 - 3.5
NOEC (24h)=0.32 mg/l
LOEC (24h) =0.56 mg/l


EC50 (48h)=0.50 mg/l 95% confidence limits in mg/l : 0.43 - 0.58
NOEC (48h)=0.32 mg/l
LOEC (48h) =0.56 mg/l

The NOEC is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item*

Reported statistics and error estimates:

The EC50 values at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression.

The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) at 24 and 48 hours were calculated using either the Step-down Cochran-Armitage Test or Step-down Rao-Scott-Cochran-Armitage test incorporating Tarone’s test and Qualitative Trend Analysis by Contrasts. All results were calculated using the ToxRat Professional computer software package (TOXRAT, 2001-2018).

Due to the nature of the data it was not possible to determine 95% confidence limits for the 48-Hour EC50 value.

Immobilization Data

Cumulative Immobilization Data and Observations in the Range-finding Test

	Observations (initial population: 5 Per replicate
	24 Hours				48 hours
	Cumulative immobilized Daphnia		Observations		Cumulative immobilized Daphnia		Observations
Nominal concentration (mg/l)	R1	R2	R1	R2	R1	R2	R1	R2
Control	0	0	5N	5N	0	0	5N	5N
0.1	0	0	5N	5N	0	0	5N	5N
1.0	5	4	AI	1N	5	5	AI	AI
10	5	5	AI	AI	5	5	AI	AI
100	5	5	AI	AI	5	5	AI	AI

R = Replicate N = Normal AI = All Daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

	24 Hours
Geometric Mean Measured Test Concentration (mg/l)	Cumulative immobilized Daphnia (Initial Population: 5 per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	5N	5N
0.042	0	0	0	0	0	0	5N	5N	5N	5N
0.085	0	0	0	0	0	0	5N	5N	5N	5N
0.14	0	0	0	0	0	0	4N 1T	5N	5N	5N
0.36	3	0	1	3	7	35	2N	4N 1RM	3N 1RM	1N 1RM
0.80	5	3	1	1	10	50	AI	2RM	2T&RM 2RM	3N 1 RM

	48 Hours
Geometric Mean Measured Test Concentration (mg/l)	Cumulative immobilized Daphnia (Initial Population: 5 per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	5N	3N 2T
0.042	0	0	0	0	0	0	5N	5N	5N	4N 1T
0.085	0	0	0	0	0	0	5N	5T	2N 3T	5N
0.14	0	0	0	0	0	0	4N 1T	4N 1T	5N	1N 4T
0.36	5	0	3	5	13	65	AI	2T 1N 2RM	2N	AI
0.80	5	5	5	5	20	100	AI	AI	AI	AI

R = Replicate N = Normal RM = Reduced mobility T = Trapped, at 24 hours any trapped Daphnia were resubmerged AI = All Daphnia immobilized

Validation Criteria

The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria

Temperature was maintained at approximately 19 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 906 to 973 Lux.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Validity criteria fulfilled:

yes

Conclusions:

The EC50, NOEC and LOEC in an acute toxicity to Daphnia magna was found to be 0.34 mg/l; 0.14 mg/l and 0.36 mg/l respectively.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test item at nominal concentrations of 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L for 48 hours at a temperature of approximately 19 C to 21 C under semi-static test conditions. The number of immobilized Daphnia was recorded after 24 and 48 hours.

Analysis of the freshly prepared test solutions at 0 and 24 hours showed measured test concentrations to range from 89% to 105% of nominal. Analysis of the corresponding aged test solutions at 24 and 48 hours showed a concentration dependent decline in measured test concentrations in the range of 18% to 76% of nominal indicating 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 0.042, 0.085, 0.14, 0.36 and 0.80 mg/L. Exposure of Daphnia magna to the test item gave the following results:

Time point (hours)	EC50 (mg/l)	95% confidence limits (mg/l)	NOEC in mg/l	LOEC in mg/l
48	0.34	not determined due to nature of data	0.14	0.36

Description of key information

The EC50, NOEC and LOEC in an acute toxicity to Daphnia magna was found to be 0.34 mg/l; 0.14 mg/l and 0.36 mg/l respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

0.34 mg/L