N,N'-methylenedistearamide

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:

28 Aug 2018 to 31 Oct 2018

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (275 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (the first approximate 2 liters used to pre condition the filter was discarded), to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnid
- Age of test organisms at initiation: < 24 hours. 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.
- Feeding during test: No

CULTURING
- Culturing conditions: 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. Culture conditions ensured that reproduction was by parthenogenesis.
- Feeding: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and GEMMA Micro 300 suspension. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 10, 18, 32, 56 and and 100 % (v/v) saturated solution.
- Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers were used.
- Type: Closed; the test vessels were then covered to reduce evaporation
- Fill volume: Approximately 100 mL
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 medium

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: 896 to 1004 Lux. The light intensity during the light period was measured using an ATP Instrumentation Lux meter.

WATER QUALITY MEASUREMENTS
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

EFFECT PARAMETERS MEASURED: immobilization
Any immobilization or adverse reactions (sub-lethal effects) to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnids were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Methods: Two range finding tests were performed. The first test was invalid due to the numbers of immobilized daphnids observed in the control (>10% immobilized) and at each test level (between 30 and 40% irrespective of concentration); therefore, the results of this range finding test have not been used for reporting purposes. In the second range finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution. The preparation of the test concentrations and exposure conditions in the second range finding test were the same as those used in the first test. A nominal amount of test item (275 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (the first approximate 2 liters used to pre condition the filter was discarded), to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. In the second range finding test five daphnids were placed in each test and control vessel and the vessels were placed in a temperature-controlled room, which maintained the water temperature at 18 to 22 °C with a maximum deviation of ± 1°C and a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours, with 20 minute dawn and dusk transition periods. Two replicate vessels were prepared for the control and at each test level. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation.
The control group was maintained under identical conditions but not exposed to the test item. After 24 and 48 hours the numbers of immobilized daphnids were recorded. A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only the concentrations to be used in the definitive test were analyzed.
- Results: No significant immobilization (≤10%) was observed at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution; however, 90% immobilization was observed at 100% v/v saturated solution. However, a similar pattern of immobility was not observed in the definitive test suggesting that the effects exhibited in the range finder were not attributable to the test item. No sub-lethal effects of exposure were observed throughout the test. Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test. Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the LOQ of the analytical method employed. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Reference substance (positive control):

yes

Remarks:

potassium dichromate. The positive control was conducted from 25 September 2018 to 27 September 2018.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution (v/v)

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: No statistically significant effects observed. The EC50 was determined as greater than the measured saturation (<0.0011 mg/L).

Details on results:

The results are tabulated in 'Any other information on results incl. tables'.
- Immobilization: There were no significant levels of immobilization (≤10%) in the groups of 20 daphnids exposed at 10 to 100% v/v saturated solution for a period of 48 hours. One daphnid (5%) in the 32 and 56% v/v saturated solution treatment group was immobilized. Two daphnids (10%) in the 100% v/v saturated solution were immobilized.
- Sub-lethal effects: No sub lethal effects of exposure were observed throughout the test

Results with reference substance (positive control):

The 24-h and 48-h EC50 values of the positive control potassium dichromate were determined to be 1.1 and 0.80 mg/L, respectively. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

The ToxRat programme was used where necessary.

Table: Cumulative Immobilization Data and Observations in the Definitive Test after 24 hours

Nominal Concentration (% v/v Saturated Solution)	24 Hours
	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	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	0	0	0	0	5 N	5 N	5 N	5 N
56	0	0	1	0	1	5	5 N	5 N	4 N	5 N
100	0	0	0	0	0	0	5N	5 N	5 N	5 N

R = Replicate

N = Number of daphnids appearing normal

Table: Cumulative Immobilization Data and Observations in the Definitive Test after 48 hours

Nominal Concentration (% v/v Saturated Solution)	48 Hours
	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	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	1	0	0	0	1	5	4 N	5 N	5 N	5 N
56	0	0	1	0	1	5	5 N	5 N	4 N	5 N
100	0	0	0	2	2	10	5 N	5 N	5 N	3 N

R = Replicate

N = Number of daphnids appearing normal

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'

Conclusions:

Based on these findings, the 48-h EC50 value for was derived to be >100% v/v of a saturated aqueous solution. There were no statistically significant toxic effects at saturation (<0.0011 mg/L).

Executive summary:

The acute toxicity to aquatic invertebrates was determined in a study according to OECD 202 and in compliance with GLP criteria. In this study daphnids (D. magna, 20 per concentration) were exposed in to nominal saturated solutions of the test substance of 0, (control), 10, 18, 32, 56 and 100% (v/v) for 48 hours under static conditions. These concentrations were selected based on an initial dose range finding study. Analytical confirmation showed that the measured test concentrations ranged from less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.0011 mg/L) to 0.00305 mg/L except for two samples where measured levels of 0.0126 and 0.0149 mg/L were determined.  Where concentrations were below the LOQ, this does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ. Given this it was considered appropriate to calculate results based on the nominal test concentration (100% v/v saturated solution). Immobilization and sub-lethal effects were recorded after 24 and 48 hours exposure. There were no significant levels of immobilization or sub-lethal effects (≤10%) in the groups of 20 daphnids exposed at 10 to 100% v/v saturated solution. Based on these findings, the 48-h EC50 value was derived to be > 100% v/v of a saturated aqueous solution. There were no statistically significant toxic effects at saturation (<0.0011 mg/L).

Description of key information

48-h EC50: >100% (v/v) of a saturated aqueous solution (Daphnia magna).