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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 Jun 2017 to 07 Sep 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study is conducted to support notification works in other worldwide jurisdictions. No proposal is required to conduct this test.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

OECD Guideline 211. Daphnia magna, Reproduction Test, 2012.

Deviations:

yes

Remarks:

see "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the control.
Sampling:
Frequency: At the beginning and at the end of four intervals of 24 hours (nominal days 0 and 1, 8 and 9, 15 and 16, 20 and 21). In addition, samples were taken from the freshly prepared stock solution at nominal days 0, 8, 15 and 20.
Volume: 1.0 mL
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the refreshment period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The batch of MLA-3202 (Naugalube OFM 3202) tested was a clear amber-red liquid and a UVCB substance. The test item was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test substance.
Preparation of test solutions started with a stock solution of 1000 μg/L applying a one-hour period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

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: To initiate the test, young daphnids < 24 hours old 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 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22 °C
Feeding: Daily, a suspension of fresh water algae
Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.
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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Total hardness varied between 143 and 161 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the study plan (>140 mg CaCO3 per liter).

Test temperature:

The temperatures in the test media varied between 19 and 20°C. The temperature continuously measured in a temperature control vessel was maintained 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).

pH:

The pH remained within the range of 7.4 to 8.4 throughout the test and thus was maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).

Dissolved oxygen:

The oxygen concentration in all test solutions remained within the range of 7.7 to 9.7 mg/L during the exposure period and thus complied with the requirements as laid down in the study plan (> 3 mg/L).

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

MLA-3202 (Naugalube OFM 3202) 6.0, 14, 35, 83 and 200 μg/L

Details on test conditions:

Acute Toxicity
According to data from an acute toxicity test, the 48h-EC50 was 0.14 mg/L based on average exposure concentrations (95% confidence interval between 0.12 and 0.17 mg/L) (Project 511881).

Range-Finding Test
A preliminary test was performed prior to the reproduction test. The concentrations of MLA-3202 (Naugalube OFM 3202) tested were: 2.0, 20 and 200 μg/L. A control group was also included. Each concentration consisted of two replicates containing a total of ten daphnids (five each). The total test period was ten days. Test conditions were held as similar as possible to those applied in the reproduction test including feeding. Test solutions were renewed on days 1, 3 and 6 during the test. Samples for possible analysis were taken on days 0 (fresh), 1 (fresh and old), 3 (fresh and old) and 6 (old). Samples were taken from solutions containing the daphnia and food (algae) and also from an extra vessel incubated under the same conditions, but without daphnia and algae. In addition, samples were taken from the stock solution at the start of each refreshment period.

Reproduction Test
Test Procedure and Conditions
Test duration: 21 days
Test type: Semi-static
Frequency of renewal: Daily
Test vessels Volume: 60 ml (6 x Ø 3.5 cm), all-glass, covered with a Perspex plate. Test vessels were incubated with test solutions for approximately 24 hours prior to exposure.
Medium: M7
Experimental design: At the start of the experiment (nominal day 0) 10 neonate daphnids, less than one day old, per group were divided over ten vessels each containing a minimum of 50 ml test medium. The control group consisted of 20 daphnids.
Light: 16 h photoperiod daily;
intensity at the start: 685 - 720 lux
intensity at the end: 700 - 785 lux
Feeding: Twice daily an amount of 0.25 mL of a Chlorella sp. suspension. On weekend days an amount of 0.50 mL was added in one single feed. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211.

Measurements and Recordings
Parental daphnids
Condition: Daily, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Daily.
Body length: At the end of the test.
Offspring
Appearance first brood: When observed.
Newborn daphnids: Daily, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.
Test medium
Temperature, oxygen and pH: At the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids.
Hardness: Once a week in fresh and old media from the control and the highest test concentration
Light: At the start and the end of the test

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

33 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction, growth and survival

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 33 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Range-Finding Test
There was no mortality of parental daphnids observed during this test.
The first brood was observed on day 8 of exposure in all groups tested. No significant differences in the number of offspring were observed in this test between the control and test groups.
One immobile offspring was observed in 20 μg/L. No incidence of aborted eggs was recorded in this test.
Samples taken from nominal 200 μg/L and the freshly prepared stock solutions were analysed. The actual concentrations measured in freshly prepared solutions were at the level of nominal (i.e. 91-105% of the nominal concentration). The measured concentrations decreased by more than 86% during the different renewal periods regardless of the presence of algae. This indicates that the decrease of test concentrations might be due to adsorption to glass. Therefore, the vessels used for the final test were pre-incubated with the respective test solutions prior to exposure.
Test conditions during the range-finding test were maintained within the limits prescribed by the study plan.

Reproduction test attempt (flow-through)
A final reproduction test attempt was performed using a flow-through system. A stock solution of 2.0 g/L was prepared in Dimethylformamide (DMF, Merck, Darmstadt, Germany). Target concentrations between 6.0 and 200 μg/L were prepared by diluting the stock solution in M7 medium. However, analyses on two successive days before the intended study start proved that the target concentrations could not be reached. Recoveries relative to the targets generally ranged between 3.8 and 54% with quite some variation between the various test groups. Stock solutions were also analysed. The actual concentrations were at the level of 105-117% of nominal and confirmed proper preparation. The project was consequently continued with a semi-static final test design with daily renewals.

Reproduction Test
Measured Concentrations
The concentrations measured in freshly prepared solutions was at the level of nominal (81-112% of nominal). The actual concentrations in the spent solutions were in most cases below 4.0 μg/L despite the fact that the test vessels had been pre-incubated with the respective test solutions before the exposure period. Based on these results the average exposure concentrations were calculated.
A response was measured in the control treatment during the last refreshment period.
Therefore, the reserve sample taken from the freshly prepared solution was analysed. This sample proved to be clean indicating that the test solutions were not contaminated. No reserve sample for the spent solution was available for analysis. However, since in all other test groups the measured concentrations were below 4.0 μg/L, it was assumed that there was no contamination of the control treatment.

Condition of Parental Daphnids
Mortality in the control reached 10% and thus complied with the requirement of the guideline. At the different groups tested, the mortality varied between 0 and 20% and was not related to the applied dose or statistically different from the control treatment. Therefore, according to the guideline, the effect parameters were calculated based on the surviving animals.

Time to First Reproduction
There was no significant delay in the appearance of the first brood between the treatments and the control during the test.

Reproduction
On average, 163 offspring were produced per surviving daphnid in the control treatment. At the different test concentrations, the mean reproduction was between 159 and 172 offspring per surviving parent and was statistically not different from the control treatment.
A significant number of aborted eggs was observed in the control treatment, whereas low numbers were recorded in the test concentrations. Most of the aborted eggs in the control (36 of 42), were recorded at the end of the test in one replicate. The reason for this is unknown.

Body Length
The growth of daphnids, as measured by the length of the surviving parents at the end of the exposure, was not significantly affected by any of the treatments.

Experimental Conditions
The pH remained within the range of 7.4 to 8.4 throughout the test and thus was maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).
The oxygen concentration in all test solutions remained within the range of 7.7 to 9.7 mg/L during the exposure period and thus complied with the requirements as laid down in the study plan (> 3 mg/L).
The temperatures in the test media varied between 19 and 20 °C. The temperature continuously measured in a temperature control vessel was maintained 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).
Total hardness varied between 143 and 161 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the study plan (>140 mg CaCO3 per liter).
The total dissolved organic carbon content of the M7 medium was 0.046 mg/L. This complied with the requirements as laid down in the guidelines (TOC < 2 mg/L).

Results with reference substance (positive control):

Not specified

Reported statistics and error estimates:

Multiple testing to find the NOEC- the SD Cochran-Armitage was performed
To justify the use of the Step-down Cochran-Armitage test at first a trend analysis by contrasts using proportions was performed.

Qualitative Trend Analysis by Contrasts (Monotonicity of Concentration/Response)
Qualitative trend analysis by contrasts (monotonicity of concentration/response) with immobility at 21 d: Psi: total of proportions weighted by contrasts; Var(psi): variance of psi; df: degrees of freedom; Chi²: Chi²-statistic; p(Chi²): probability that the trend is due to chance (Ho: Slope = 0).
Hypothesis of monotonicity is accepted if at least the linear contrast is significant.

The linear trend is not significant (p > 0.05)
The quadratic trend is not significant (p > 0.05)
The analysis of contrasts did not reveal a linear trend, thus the selected Step-down Cochran-Armitage test was replaced by the Bonferroni Fisher test.

Fisher`s Exact Binomial Test with Bonferroni Correction
Fisher´s Exact Binomial Test with Bonferroni Correction with immobility at 21 d: Two-sample comparisons between treatment and control on the multiple significance level (Alpha is 0.050; one-sided greater). Two-sample comparisons are performed sequentially using the adjusted Alpha* (= alpha/(k-1); k: number of comparisons (after Holm, 1979); Ho (no effect) is accepted, if the probability p > Alpha*; p(exact) is the probability that the increase in category "Immobile" observed in the treatments is due to chance. Note that the step-down test terminates after the first non-significant treatment is encountered

Mortality and Reproductive Potency During the Range-Finding Test

Day	MLA-3202 (Naugalube PFM 3203), Nominal concentration (μg/L)
	Control		2.0		20		200
	A	B	A	B	A	B	A	B
8 9 10	36 3 0	29 6 0	24 6 0	23 3 0	35 1, 1i 0	23 4 0	30 0 0	33 0 0
Total Average per group	39	35 37	30	26 28	36	27 32	30	33 32
Number of survived parental daphnids	5	5	5	5	5	5	5	5

i – number of immobile offspring

Nominal and Mean Measured Exposure Concentrations

MLA-3202 (Naugalube OFM 3202) Nominal concentration (mg/L)	Mean exposure concentration (μg/L)				Average exposure concentration (μg/L)
	Day 0-1	Day 8-9	Day 15-16	Day 20-21
6.0 14 35 83 200	3.1 4.8 7.9 12 71	3.3 5.0 8.4 13 21	3.7 5.5 19 13 20	3.5 5.3 8.2 13 19	3.4 5.1 11 13 33

 

Mortality (Immobility) of Parental Daphnids at the End of the Test

MLA-3202 (Naugalube OFM 3202) Average exposure conc. (μg/L)	Introduced	Mobile	Immobile	%Immobility
Control 3.4 5.1 11 13 33	20 10 10 10 10 10	18 8 10 10 8 9	2 2 0 0 2 1	10 20 0 0 20 10

 

Group Mean Cumulative Number of Juveniles per Surviving Parent and Reduction of Reproduction at the End of the Test

MLA-3202 (Naugalube OFM 3202) Average exposure conc. (μg/L)	Mean	Std. Dev.	n	%Reduction
Control 3.4 5.1 11 13 33	163 159 171 172 169 160	14 21 12 13 8.7 43	18 8 10 10 8 9	2.4 -4.7 -5.5 -3.9 1.9

 

Incidence of Immobilised Offspring, Offspring Trapped at the Surface and Aborted Eggs

Abnormality	MLA-3202 (Naugalube OFM 3202); Average exposure conc. (μg/L)
	Control	3.4	5.1	11	13	33
Immobile offspring Aborted eggs	3 42	2 1	2 2	4 3	1 6	7 1

 

Group Mean Body Lengths (mm) and Reduction of Growth of Parental Daphnids at the End of the Test

MLA-3203 (Naugalube OFM 3202) Average exposure conc. (μg/L)	Mean (mm)	Std. Dev.	n	%Reduction
Control 3.4 5.1 11 13 33	4.51 4.68 4.52 4.54 4.59 4.49	0.204 0.102 0.118 0.094 0.059 0.256	18 8 10 10 8 9	-3.7 -0.2 -0.7 -1.7 0.5

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study MLA-3202 (Naugalube OFM 3202) did not affect reproduction, growth and survival of Daphnia magna at 33 μg/L after 21 days of exposure. The NOEC for MLA-3202 (Naugalube OFM 3202) was 33 μg/L and the 21-day EC50 for reproduction was >33 μg/L.

Executive summary:

This study is conducted to support notification works in other worldwide jurisdictions. No proposal is required to conduct this test.

The objective of the test was to evaluate the effects of MLA-3202 (Naugalube OFM 3202) on the survival, growth and reproductive capacity of the cladoceran, Daphnia magna during a 21-day exposure period under semi-static test conditions. For this purpose, test organisms were exposed to aqueous solutions containing the test item at various concentrations. The time of the first production of young, the number of young born, immobility and other signs of intoxication were observed and compared with corresponding parameters in the controls.

 

The study procedures described in this report were based on the OECD guideline for Testing of Chemicals: Guideline No. 211, 2012.

 

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

 

The reproduction test was performed in a semi-static system and was comprised of 10 vessels per test concentration and 20 vessels for an untreated control group. At the start of the test, each of the vessels contained one neonate (<24h old) Daphnia magna in 50 ml test medium.

The nominal concentrations of MLA-3202 (Naugalube OFM 3202) tested were 6.0, 14, 35, 83 and 200 μg/L. The study duration was 21 days and the test solutions were renewed daily.

The daphnids were fed on a daily basis with a Chlorella pyrenoidosa suspension. Every day the condition of the parental daphnids was recorded. During the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test, the body lengths of the surviving parental daphnids were measured.

 

During the study, samples for analyses were taken at the beginning and the end of four intervals of 24 hours.

 

Samples taken from all groups were analysed. The concentrations measured in freshly prepared solutions were at the level of nominal (81-112% of nominal). The actual concentrations in the spent solutions were in most of the cases below 4.0 μg/L. Based on these results the average exposure concentrations were calculated to correspond with 3.4, 5.1, 11, 13 and 33 μg/L in nominally 6.0, 14, 35, 83 and 200 μg/L, respectively.

 

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

 

Mortality in the control reached 10% and thus complied with the requirement of the guideline. At the different groups tested the mortality varied between 0 and 20% and was not related to the applied dose or statistically significant from the control treatment. Therefore, according to the guideline, the effect parameters were calculated based on the surviving animals.

 

The average cumulative number of young per female in the control after 21 days was 163 ± 8.9 (mean ± standard deviation).

 

No significant differences were found for reproduction, age at first reproduction and growth (expressed as the length of surviving parental daphnids) between the control and any of the concentrations tested.

 

Effect parameters (mg/L) obtained in this study are summarized in the table below

Parameter	MLA-3202 (Naugalube OFM 3202) Average exposure conc. (μg/L)
NOEC for reproduction	33
EC10 for reproduction	>33*
EC50 for reproduction	>33*
NOEC for mortality	33
NOEC for growth	33

* - 95% confidence intervals could not be given

 

In conclusion, MLA-3202 (Naugalube OFM 3202) did not affect reproduction, survival and growth of Daphnia magna exposed to concentrations up to including 33 μg/L for 21 days.

The NOEC for MLA-3202 (Naugalube OFM 3202) was 33 μg/L and the 21-day EC50 for reproduction was >33 μg/L.

Description of key information

This study is conducted to support notification works in other worldwide jurisdictions. No proposal is required to conduct this test.

MLA-3202 (Naugalube OFM 3202) did not affect reproduction, survival and growth of Daphnia magna exposed to concentrations up to including 33 μg/L for 21 days.

The NOEC for MLA-3202 (Naugalube OFM 3202) was 33 μg/L and the 21-day EC50 for reproduction was >33 μg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

33 µg/L

Additional information

The objective of the test was to evaluate the effects of MLA-3202 (Naugalube OFM 3202) on the survival, growth and reproductive capacity of the cladoceran, Daphnia magna during a 21-day exposure period under semi-static test conditions. For this purpose, test organisms were exposed to aqueous solutions containing the test item at various concentrations. The time of the first production of young, the number of young born, immobility and other signs of intoxication were observed and compared with corresponding parameters in the controls.

 

The reproduction test was performed in a semi-static system and was comprised of 10 vessels per test concentration and 20 vessels for an untreated control group. At the start of the test, each of the vessels contained one neonate (<24h old) Daphnia magna in 50 ml test medium.

The nominal concentrations of MLA-3202 (Naugalube OFM 3202) tested were 6.0, 14, 35, 83 and 200 μg/L. The study duration was 21 days and the test solutions were renewed daily.

The daphnids were fed on a daily basis with a Chlorella pyrenoidosa suspension. Every day the condition of the parental daphnids was recorded. During the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test, the body lengths of the surviving parental daphnids were measured.

 

During the study, samples for analyses were taken at the beginning and the end of four intervals of 24 hours.

 

Samples taken from all groups were analysed. The concentrations measured in freshly prepared solutions were at the level of nominal (81-112% of nominal). The actual concentrations in the spent solutions were in most of the cases below 4.0 μg/L. Based on these results the average exposure concentrations were calculated to correspond with 3.4, 5.1, 11, 13 and 33 μg/L in nominally 6.0, 14, 35, 83 and 200 μg/L, respectively.

 

Mortality in the control reached 10% and thus complied with the requirement of the guideline. At the different groups tested the mortality varied between 0 and 20% and was not related to the applied dose or statistically significant from the control treatment. Therefore, according to the guideline, the effect parameters were calculated based on the surviving animals.

 

The average cumulative number of young per female in the control after 21 days was 163 ± 8.9 (mean ± standard deviation).

 

No significant differences were found for reproduction, age at first reproduction and growth (expressed as the length of surviving parental daphnids) between the control and any of the concentrations tested.

 

Effect parameters (mg/L) obtained in this study are summarized in the table below

Parameter	MLA-3202 (Naugalube OFM 3202) Average exposure conc. (μg/L)
NOEC for reproduction	33
EC10 for reproduction	>33*
EC50 for reproduction	>33*
NOEC for mortality	33
NOEC for growth	33

* - 95% confidence intervals could not be given

 

In conclusion, MLA-3202 (Naugalube OFM 3202) did not affect reproduction, survival and growth of Daphnia magna exposed to concentrations up to including 33 μg/L for 21 days.

The NOEC for MLA-3202 (Naugalube OFM 3202) was 33 μg/L and the 21-day EC50 for reproduction was >33 μg/L.