Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

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:

21 November 2016 to 26 January 2017

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:

the Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004.

Deviations:

yes

Remarks:

for details see 'Any other information on materials and methods incl. tables'

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Council Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.2. "Daphnia sp. Acute Immobilisation Test".

Deviations:

yes

Remarks:

for details see 'Any other information on materials and methods incl. tables'

GLP compliance:

yes

Specific details on test material used for the study:

Test item: 207258/AIdentification: MLA-3202Appearance: Clear amber-red liquidBatch: RC-1045Purity/Composition: UVCBTest item storage: At room temperature Stable under storage conditions until: 17 February 2019 (expiry date)Purity/composition correction factor: No correction factor requiredChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3

Analytical monitoring:

yes

Details on sampling:

Test samples were stored in the freezer (≤ -15°C). Storage stability of samples under these conditions was demonstrated in project 511870.On the day of analysis, the test samples were thawed at room temperature. The samples were diluted in a 1:3 (v:v) ratio with methanol and analyzed. If necessary, the samples were further diluted with 75/25 (v/v) methanol/ISO-medium to obtain concentrations within the calibration range.Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below. Frequency: at t=0 h and t=48 hVolume: 1.0 mL from the approximate centre of the test vessels Storage : Samples were stored in a freezer (≤ -15ºC) until analysis.At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additionally, single reserve samples of 1.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤ -15ºC) for possible analysis until delivery of the final report with a maximum of three months.Immobility (including mortality): At 24 hours and at 48 hours.pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Vehicle:

yes

Details on test solutions:

The batch of MLA-3202 tested was a clear amber-red liquid completely soluble in test medium at the concentrations tested. The test item was a UVCB substance. No correction was made for the purity/composition of the test item.Preparation of test solutions started with the highest concentration of 1 mg/L applying one hour of magnetic stirring to accelerate the dissolution of the test item in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All final test solutions were clear and colourless.Note that in the combined limit/range-finding test the highest test concentration was 100 mg/L and was stirred for a period of 2.5 hours. The highest concentration in this test was hazy while the remaining concentrations were clear and colorless. In the range-finding test, the highest test concentration was 10 mg/L. All solutions were clear and colorless.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.Source: In-house laboratory culture with a known history.Reason for selection: This system has been selected as an internationally accepted invertebrate species.Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.Characteristics: For the test, young daphnids with an age of < 24 hours were selected from parental daphnids older than two weeks.Breeding Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.Maximum age of the cultures: 4 weeksRenewal of the cultures: After 7 days of cultivation half of the medium twice a week.Temperature of medium: 18-22°CFeeding: Daily, a suspension of fresh water algae.Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test type:

static

Water media type:

other: Adjusted ISO medium (M7, as prescribed by Dr. Elendt-Schneider)

Remarks:

see 'Any other information on materials and menthods incl. tables' for more details.

Limit test:

yes

Total exposure duration:

48 h

Hardness:

180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.

Test temperature:

18 22°C, constant within 2°C

pH:

6.0-9.0

Dissolved oxygen:

≥ 3 mg/L

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Combined limit/range-finding test0.10, 1.0 and 10 mg/LLimit test 0.10 to 10 mg/L increasing by a factor of 10 and to a control. Final test MLA-32020.10, 0.18, 0.32, 0.56 and 1.0 mg/LControl: Test medium without test item or other additives.

Details on test conditions:

Test duration: 48 hoursTest type:StaticTest vessels: 100 mL, all-glass Medium:Adjusted ISO medium Number of daphnids:20 per concentrationLoading:5 per vessel containing 80 mL of test solutionLight:16 hours photoperiod dailyFeeding:No feeding Aeration:No aeration of the test solutions.Introduction of daphnids: Within 36 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval between 0.22-0.31 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval between 0.12 and 0.17 mg/L

Details on results:

Combined limit/range-finding testAt the end of the exposure period, more than 10% of the daphnids were observed to be immobile in the control group, therefore the test was considered not valid. Nevertheless, the responses were used to set the range of concentrations to be tested in the range-finding test. No immobility was observed at the lowest test concentration throughout the test duration, while effects were complete already after 24 hours of exposure at the three highest test concentrations. Samples taken from nominally 0.10 and 1.0 mg/L were analysed. The actual concentrations were 0.10 and 1.1 mg/L at the start of the test, respectively. These concentrations decreased to 23-74% of initial at the end of the test. All test conditions were maintained within the limits prescribed by the study plan.Range-finding testThe highest concentration tested in the range-finding test was 10 mg/L. This concentration was clear and colorless at the end of the preparation period unlike the concentration of 100 mg/L prepared in the preceding test. No immobility was observed in the control and at the lowest test concentration during the test period, while effects were complete already after 24 hours at the two higher test concentrations. Therefore, the 48h-EC50 was expected between 0.10 and 1.0 mg/L.Microscopic observations of daphnids exposed to 1.0 mg/L (after 24h and 48h of exposure) and 10 mg/L (after 48h of exposure) revealed no test item attached to their bodies.All test conditions were maintained within the limits prescribed by the study plan.Final testMeasured concentrationsSamples taken from all test concentrations were analysed. Measured concentrations were at the level of nominal at the start of the test (85-91%) and decreased to 10-84% of initial at the end of the exposure period. Therefore, the average exposure concentrations were calculated and used to determinate the effect parametersImmobilityNo immobility was observed in the control and at the lowest test concentration, while effects were complete already after 24 hours of exposure at the highest test concentration. After 24 hours of exposure, no daphnids were immobile at 0.10 mg/L while 35 and 90% were immobile at 0.22 and 0.44 mg/L, respectively. At the end of the exposure period, 20% and 85% of daphnids were immobile at 0.10 and 0.22 mg/L, respectively, while at the two highest concentrations the effects were complete.The responses recorded in this test allowed for reliable determination of an EC50. Experimental conditionsThese test conditions remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: ≥ 3 mg/L at the end of the test).The temperature continuously measured in a temperature control vessel remained stable at 20°C during the test, and complied with the requirements as laid down in the study plan (18 - 22°C, constant within 2°C).

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was within the expected range of 0.28 to 0.9 mg/L. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at Charles River Den Bosch.The 24h-EC50 was 0.91 mg/L with a 95% confidence interval ranging from 0.77 to 1.1 mg/L. The 48h-EC50 was 0.58 mg/L with a 95% confidence interval ranging from 0.49 to 0.67 mg/L.

Reported statistics and error estimates:

Not specified

Calibration Curves

Calibration curves were constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.

QC Samples

A small response at the retention time of the test item was detected in the chromatograms of the blank QC samples. Concentration was below the limit of detection which was 0.00029 mg/L during the range-finding test and 0.00012 mg/L during the final test.

During the range-finding test, the mean accuracy of the 0.01 mg/L QC samples was slightly above the criterion range of 70 -110% (i.e. 111% of target). The mean accuracy of the 10 mg/L QC samples was within the criterion range. During the final test, the mean accuracies of QC samples containing test item fell within the criterion of 70-110%. It demonstrated that the analytical method was adequate for the determination of the test item concentration in the test samples.

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 was 0.14 mg/L based on average exposure concentrations (95% confidence interval between 0.12 and 0.17 mg/L).

Executive summary:

An Acute Toxicity Study (Static) using MLA-3202 on Daphnia magna was conducted according to OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008.

The batch of MLA-3202 tested was a clear amber-red liquid completely soluble in test medium at the concentrations tested. The test item was a UVCB substance.

 

A full test was performed based on the results of a combined limit/range-finding test and a range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

Samples taken from all test concentrations were analyzed. Measured concentrations were at the level of nominal at the start of the test (85-91%) and decreased to 10-84% of initial at the end of the exposure period.Based on these results, the average exposure concentrations were 0.029, 0.10, 0.22, 0.44 and 0.81 mg/L in nominally 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L, respectively.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The 48h-EC50 was 0.14 mg/L based on average exposure concentrations (95% confidence interval between 0.12 and 0.17 mg/L).

Description of key information

Key value determined in GLP accredited laboratory study according to the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No 440/2008, Part C.2, 2008.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.14 mg/L

Additional information

A full test was performed based on the results of a combined limit/range-finding test and a range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

Samples taken from all test concentrations were analyzed. Measured concentrations were at the level of nominal at the start of the test (85-91%) and decreased to 10-84% of initial at the end of the exposure period.Based on these results, the average exposure concentrations were 0.029, 0.10, 0.22, 0.44 and 0.81 mg/L in nominally 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L, respectively.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The 48h-EC50was 0.14 mg/L based on average exposure concentrations (95% confidence interval between 0.12 and 0.17 mg/L).