Poly(oxy-1,2-ethanediyl), alpha-[2-[bis(2-aminoethyl)methylammonion)ethyl]-, omega-hydroxy, N,N'-di-Limnanthes alba seed oil acyl derivatives, methyl sulfates (salts)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2017 - May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study to GLP standards with no deviations from the study plan

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Poly(oxy-1,2-ethanediyl), alpha-[2-[bis(2-aminoethyl)methylammonion)ethyl]-, omega-hydroxy, N,N'-di-Limnanthes alba seed oil acyl derivatives, methyl sulfates (salts)

EC Number:

629-704-1

Cas Number:

226995-92-2

Molecular formula:

The substance is a UVCB - no specific molecular weight or formula is available.

IUPAC Name:

Poly(oxy-1,2-ethanediyl), alpha-[2-[bis(2-aminoethyl)methylammonion)ethyl]-, omega-hydroxy, N,N'-di-Limnanthes alba seed oil acyl derivatives, methyl sulfates (salts)

Test material form:

liquid

Specific details on test material used for the study:

Identification: RM1004942
Batch: RL149/17
Purity: 100% UVCB
Physical state/Appearance: Amber colored paste
Expiry Date: 08 September 2018
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 and 24 hours and from pooled replicates at 24 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis, if necessary. Only samples at the NOEL rate and above were analyzed as it was expected that the analytical method would not be sufficiently sensitive to achieve values at the lower concentrations.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a water accommodated fraction (WAF) of the test item.

The loading rates to be used in the initial experiments and definitive test were determined by a preliminary range-finding test.

Test solutions

Vehicle:

no

Details on test solutions:

RANGE-FINDING TEST
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/l. Nominal amounts of test item (5, 50 and 500 mg) were each separately added to a glass slide and suspended in the water column of 5 liters of test water to give the 1.0, 10 and 100 mg/l loading rates, respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water columns and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed microdispersions to be present, therefore the WAF was filtered through a sheet of filter paper. Following filtration through the filter paper, some test item microdispersions were still present however, it was considered unlikely that any further filtration would remove more test item.

Several tests were conducted at nominal loading rates between 0.078 and 10 mg/l, however, in these tests undissolved material was observed to come through the filters causing various levels of physical effects on the Daphnia which were confounding the results. These inconsistencies in the presence of the undissolved test item in the test media gave rise to inconclusive patterns of immobilization, that were not a true representation of the toxicity of the test item.


Based on the results of the range-finding test and the initial tests the following loading rates were assigned to the definitive test: 0.16, 0.32, 0.63, 1.25 and 2.5 mg/l.

DEFINITIVE TEST
Nominal amounts of test item (6.25 and 12.5 mg) were each separately added to the surface of 5 liters of test water to give the 1.25 and 2.5 mg/l loading rates, respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 0.16, 0.32, 0.63, 1.25 and 2.5 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed potential test item to be present, therefore as a precaution, a filter paper was also used. A series of dilutions was made from the 1.25 mg/l concentration to give further test concentrations of 0.16, 0.32, 0.63 mg/l loading rate WAF.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Adult daphnids were maintained in 150 ml glass beakers containing 100 ml Elendt M7 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 (Desmodesmus subspicatus) and Tetramin® flake 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.

During the definitive study, 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 seconds after gentle agitation.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test conditions

Hardness:

no data

Test temperature:

Temperature was maintained at approximately 20 °C to 22 ºC throughout the test

pH:

7.9 to 8.1. There were no treatment related differences for pH.

Dissolved oxygen:

ranged from 8.5 to 9.6 mg/l

Salinity:

not applicable

Conductivity:

no data

Nominal and measured concentrations:

Modification of the standard method for the preparation of aqueous media was performed for this poorly water soluble substance. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

Test concentrations were measured, but reported as nominal loading rates (WAF).

Details on test conditions:

150 ml glass jars containing approximately 100 ml of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item. Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. 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.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 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Measured and reported as nominal loading rate (WAF)

Remarks:

1.5 - 3.1 CI

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Loading rate (WAF)

Details on results:

Inspection of the immobilization data at 24 hours and analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood at 48 hours based on the nominal loading rates gave the following results:

The NOEL rates after 24 and 48 hours exposure were 2.5 and 0.63 mg/l loading rate WAF, respectively. Correspondingly the LOEL rate at 48 hours was 1.25 mg/l loading rate WAF. The slope and the standard error of the response curve at 48 hours was 2.9 (standard error = 0.072).

No sub-lethal effects of exposure were observed throughout the test at concentrations of 0.16, 0.32, 0.63 and 2.5 mg/l loading rate WAF. Sub-lethal effects of exposure were observed in the test concentration of 1.25 mg/l loading rate WAF. This response was reduced mobility. Microscopic observations were conducted on the immobilized Daphnia which revealed that no undissolved test item was adhered, suggesting that physical toxicity was not a factor in this case.

Results with reference substance (positive control):

The positive control after 48-h resulted in an EC50 of 0.75 mg/l (0.56 to 1.0 mg/l CI) and a NOEC of 0.56 mg/l. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

The EL50 value and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The No Observed Effect Loading (NOEL) rate at 48 hours was calculated using the Step-down Cochran-Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT). An estimate of the EL50 value at 24 hours was given by inspection of the immobilization data.

Any other information on results incl. tables

Time Point (Hours)	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)	NOEL (mg/L)	LOEL (mg/L)
24	> 2.5	not available	2.5	-
48	2.0	1.5 to 3.1	0.63	1.25

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The OECD 202 Guideline study exposed Daphnia magna to the test substance in a semi-static test for 48-hours. The EL50 reported as a WAF loading rate was 2.0 mg/l for the mobility endpoint.

Executive summary:

A reliable study was performed to assess the acute toxicity of the test item to Daphnia magna by following 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. Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Chemical analysis of the freshly prepared 0.63, 1.25 and 2.5 mg/L loading rate WAF test preparations at 0 and 24 hours showed measured test concentrations to range from less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.37 mg/L to 0.50 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to be less than the LOQ. The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only. The 48-hour exposure to the test item resulted in an EL50 of 2.0 mg/l (WAF loading rate) (1.5 to 3.1 CI) based on an immobilisation endpoint.