Oligomerisation products of ethylene oxide with reaction products of rape oil and ethanolamine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Definitive test between 19 May 2003 and 21 May 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The following test concentrations were assigned to the definitive test: 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/l.

- Sampling method: Water samples were taken from the control (replicates RI - R2 pooled) and all the test groups (replicates RI - R2 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) for quantitative analysis.

- Sample storage conditions before analysis: Duplicate samples were taken and stored frozen (approximately -20°C) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

For the purpose of the definitive test the test material was dispersed directly in dechlorinated tap water.

An amount of test material (100 mg) was dispersed in dechlorinated tap water and the volume adjusted to I litre to give a 100 mg/l stock dispersion. An aliquot (500 ml) of this stock dispersion was dispersed in a final volume of 5 litres of dechlorinated tap water to give the 10 mg/l test concentration. To prepare the remainder of the test series of 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l, aliquots (20, 36, 64, 112, 200, 360, 640 and 1120 ml) of the 10 mg/l test concentration were each separately dispersed in a final volume of 2 litres of dechlorinated tap water.

Each stock dispersion and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The test material was weighed out and diluted under a non-actinic light source, the test vessels and sample bottles were shielded from the light and the test media was freshly prepared at 0 and 24 hours prior to dosing using a semi-static test regime.

Test organisms (species):

Daphnia magna

Details on test organisms:

Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of dechlorinated tap water in a temperature controlled room at 21°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 suspension of algae (Chlorella sp.). 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 the would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Any immobilisation 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 immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Hardness:

146 mg/l as CaCO3

Test temperature:

The test vessels were maintained in a temperature controlled room at approximately 21°C.

pH:

pH range for test samples: 7.7 - 8.1.
There were no treatment related differences for oxygen concentration or pH.

Dissolved oxygen:

Dissolved oxygen concentration expressed as a percentage of Air Saturation Value.
ASV% range for test samples: 90-96.
There were no treatment related differences for oxygen concentration.

Salinity:

Conductivity of 452 μS/cm.

Nominal and measured concentrations:

Range-finding test:
In the range-fmding test Daphnia magna were exposed to a series of nominal test concentrations of 0.1 0, 1.0, 10 and 100 mg/l. The test material was dispersed directly in water.

Definative test:
Nominal (mg/L) Time-weighted mean measured concentration (mg/l)
0.10 0.0317
0.18 0.0535
0.32 0.0802
0.56 0.152
1.0 0.193
1.8 0.312
3.2 1.13
5.6 2.20
10 4.06

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass jars
- Type (delete if not applicable): covered to reduce evaporation
- Aeration: The rest vessels were not aerated.
- Renewal rate of test solution (frequency/flow rate): For 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: 10
- No. of vessels per concentration (replicates): Duplicate vessels.
- No. of vessels per control (replicates): Duplicate vessels.


TEST MEDIUM / WATER PARAMETERS
The dechlorinated tap water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 100 mg/l as CaC03. After dechlorination and softening the water
was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

At the start of the study the diluent had a particulate matter concentration of 0.2 mg/l, total water hardness of 146 mg/l as CaCO3, alkalinity of 110 mg/l as CaCO3, conductivity of 452 μS/cmm and Total Organic Carbon content of 0.84 mg C/L


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation 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 immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: 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, 1.0, 1.8, 3.2, 5.6 and 10 mg/l.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

9.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 7.7-12 mg/l

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Results based on the time-weighted mean measured test concentrations

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Results based on the time-weighted mean measured test concentrations

Details on results:

RANGE-FINDING TEST:
No immobilisation was observed at the test concentration of 0.10 mg/l. However, immobilisation was observed at 1.0, 10 and 100 mg/I.

DEFINITIVE TEST:
Immobilisation data:

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in the table below.

Analysis of the immobilisation data at 24 and 48 hours based on the nominal test concentrations gave the following results:
24h EC50 (mg/l): 9.5
48h EC50 (mg/l): 9.5
95% Confidence limits (7.7-12 mg/l)

The No Observed Effect Concentration after 24 and 48 hours was 5.6 mg/l. The NOEC is based upon zero immobilisation at this concentration.

VERIFICATION OF TEST CONCENTRATIONS:
Analysis of the freshly prepared test concentrations at 0 and 24 hours showed the measured test concentrations to range from 87% to 114% of the nominal values. However, analysis of the old or expired test preparations sampled at 24 and 48 hours showed a marked decline in the measured test concentrations which, ranged from less than the limit of quantitation (LOQ) of the analytical method employed to 46% of the nominal values at 24 hours and after 48 hours all of the measured test concentrations were shown to be less than the LOQ which was assessed down to 0.012 mg/l.

These results were in line with the preliminary stability analyses which indicated that the test material was unstable under the test conditions employed. It was considered that biodegradation of the test material may have been a factor in the decline of the measured test concentrations over each media renewal period as the Modified C02 Evolution Test (see biodegradation section) showed 32% and 60% biodegradation after 3 and 6 days respectively, indicating the potential of the test material to biodegrade rapidly in an aqueous environment.

Given that a marked decline in measured test concentrations was observed over each 24-Hour renewal period it was considered justifiable to present a 'worst case' analysis of the data by calculating the results based on the time-weighted mean measured test concentrations also.

The time-weighted mean measured test concentrations were calculated as follows:
Nominal (mg/L) Time-weighted mean measured concentration (mg/l)
0.10 0.0317
0.18 0.0535
0.32 0.0802
0.56 0.152
1.0 0.193
1.8 0.312
3.2 1.13
5.6 2.20
10 4.06

Analysis of the immobilisation data at 24 and 48 hours based on the nominal test concentrations gave the following results:
24h EC50 (mg/l): 3.8
48h EC50 (mg/l): 3.8
95% Confidence limits (3.1 -4.8 mg/l)

The No Observed Effect Concentration after 24 and 48 hours was 2.2 mg/l. The NOEC is based upon zero immobilisation at this concentration.

The use of time-weighted mean measured concentrations was considered not to significantly alter the results of the test.

Table: Cumulative Immobilisation Data in the Definitive Test

 

Nominal concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 per replicate)
	24 hours				48 hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.10	0	0	0	0	0	0	0	0
0.18	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	0	0
5.6	0	0	0	0	0	0	0	0
10	5	6	11	55	5	6	11	55

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour ECso value of 9.5 mg/l with 95% confidence limits of 7.7 - 12 mg/l. The No Observed Effect Concentration at 48 hours was 5.6 mg/l.

Based on the time-weighted mean measured test concentrations the 48-Hour ECso was 3.8 mg/l with 95% confidence limits of 3.1-4.8 mg/l and the No Observed Effect Concentration was 2.2 mg/l.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 202, "Daphnia sp, Acute Immobilisation Test and Reproduction Test" referenced as Method C.2 of Commission Directive 92/69IEEC (which constitutes Annex V of Council Directive 67/548IEEC), US CFR Title 40 Part 797 Section 1300: US EPA Pesticide Assessment Guideline, Sub-Division E Section 72-2 and US EPA Draft Ecological Effects Test Guideline OPPTS 850.1010.

Methods.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous dispersion of the test material at ooneentrations of 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/l for 48 hours at a temperature of approximately 21°C under semi-static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

Results.

The 48-Hour EC50 for the test material to Daphnia magna based on nominal test concentrations was 9.5 mg/l with 95% confidence limits of 7.7 - 12 mg/l. The No Observed Effect Concentration was 5.6 mg/l.

Analysis of the freshly prepared test concentrations at 0 and 24 hours showed the measured test concentrations to range from 87% to 114% of the nominal values. However, analysis of the old or expired test preparations sampled at 24 and 48 hours showed a marked decline in the measured test concentrations which, ranged from less than the limit of quantitation (LOQ) of the analytical method employed to 46% of the nominal values at 24 hours and after 48 hours all of the measured test concentrations were shown to be less than the LOQ which was assessed down to 0.012 mg/l.

These results were in line with the preliminary stability analyses which indicated that the test material was unstable under the test conditions employed. It was considered that biodegradation of the test material may have been a factor in the decline of the measured test concentrations over each media renewal period as the Modified C02 Evolution Test showed 32% and 60% biodegradation after 3 and 6 days respectively, indicating the potential of the test material to biodegrade rapidly in an aqueous environment.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentrations in order to give a "worst case" analysis of the data. The 48-Hour EC50 based on the time-weighted mean measured test concentrations was 3.8 mg/l with 95% confidence limits of 3.1 - 4.8 mg/l and the No Observed Effect

Concentration was 2.2 mg/l.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 16 Setember 1991 to 26 Setember 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna, var. Straus
- Source: LWA Laboratories, Dr. Knie, W-4000 Düssel-dort, Germany
- Age at study initiation (mean and range, SD): not more than 24 hours old
- Feeding during test: Every 24 hours ad lib. with Scenedesmus cells

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20.3 ºC

pH:

7.89

Dissolved oxygen:

7.99

Details on test conditions:

TEST SYSTEM
Four test vessels involving 5 Daphnia each are run in parallel for every test concentration.
The Daphnia are exposed to various concentrations of the test substance for 48 hours. After 24h and 48h the percentage of animals incabable of swimming is recorded.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7 mg/L

	after 24 hours mg/L	after 48 hours mg/L
EC0	4	4
EC50	20	7
EC100	256	32

Validity criteria fulfilled:

yes

Conclusions:

The acute inmobilisation EC50 value of test material in Daphnia magna at 48 h exposure period was 7 mg/L.

Executive summary:

This study was performed to assess the acute immobilisation 24 h EC50, of test material in Daphnia magna. Four test vessels involving 5 Daphnia each are run in parallel for every test concentration, control, 2, 4, 8, 16, 32, 64, 128 and 256 mg/L. A static toxicity test procedure was followed. pH-value, water temperature and dissolved oxygen concentration are measured at the beginning and at the end of each test. The acute inmobilisation EC50 value of test material in Daphnia magna at 24 h exposure period was 20 mg/L and 48 h was 7 mg/L.

Description of key information

Two acute studies conducted to OECD Guideline 202. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.8 mg/L

Additional information

There are two studies available for this endpoint. The key study was chosen as the study performed under GLP-conditions (reliability 1). This study resulted in a higher toxicity of the substance and represents the worst case for acute toxicity to Daphnia. The key study gave an EC50(48h) of 9.5 mg/L (based on nominal test concentrations). The EC50 (48h) based on time-weighted mean measured test concentrations was 3.8 mg/L.

The No Observed Effect Concentration (based on time-weighted mean measured test concentrations) was 2.2 mg/l.