(S)-N,N'-dibutyl-2-[(1-oxododecyl)amino]glutaramide

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:

29 January 2018 to 31 January 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:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-2-1

Version / remarks:

(Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE).

Version / remarks:

Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Duplicate samples (from each replicates) were taken (2 x ~2 mL) in plastic tubes at the beginning and at the end of the renewal periods from the control and at the applied test concentration level.
- After sampling, samples were frozen and kept approximately at -20 °C at the Test Facility.

Vehicle:

no

Details on test solutions:

- Because the test material is very poorly soluble in water, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
- A saturated test material solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test material into the test medium (ISO Medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30 °C and then equilibrated for about 24 hours at approximately 20 °C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100 % saturated solution.
- Note that the test material dissolved very slowly, hence although the duration of formulation procedure is relatively long for a molecule which in not highly stable, it was necessary to use a long saturation solution method to achieve maximal concentration.
- The test solution was prepared just before introduction of the Daphnia (start of treatments). As a Limit test was carried out, further dilution of stock solution was not performed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Breeding: The Daphnia are bred at the testing laboratory. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
- Age: Less than 24 h old at the beginning of the test.
- Feeding during test: No

ACCLIMATION
- Acclimation period: There was no acclimatisation because the water used was similar to the culture water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

247 mg/L (as CaCO3)

Test temperature:

20.2 – 20.7 °C

pH:

7.24 – 7.71

Dissolved oxygen:

7.4 – 8.9 mg/L

Nominal and measured concentrations:

Nominal: 100 % saturated solution
Geometric mean measured concentration: 2.89 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker with a volume of 50 mL
- Material, size, headspace, fill volume: ~40 mL test solution/flask; 8 mL test solution/animal.
- Renewal rate of test solution (frequency/flow rate): The frequency of the water renewal periods was 24 hours.
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per vehicle control: 4

TEST MEDIUM / WATER PARAMETERS
- Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition was used for breeding the test animals.
- Composition of ISO medium: Separate stock solutions of individual trace elements were first prepared in deionised water at the concentrations below. The medium was prepared of these different stock solutions, so it contained all trace elements (combined solution). 25 mL/L of each stock solution was added to deionised water to prepare the media.
- CaCl2·2H2O: 11.76 g/L
- MgSO4·7H2O: 4.93 g/L
- KCl: 0.23 g/L
- NaHCO3: 2.59 g/L

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the renewal periods in each test vessel and was in the range of 7.24 – 7.71.
- Photoperiod: The test was carried out in 16-hour light and 8-hour dark cycle.
- Temperature: The water temperature was measured at the start and at the end of the renewal periods in each test vessel. The test temperature was in the range of 20.2 – 20.7 °C measured in the test vessels. The additionally measured temperature in the climate chamber was between 19.8 – 20.9 °C.
- Oxygen concentration: The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the renewal periods and was in the range of 7.4 – 8.9 mg/L.

EFFECT PARAMETERS MEASURED:
- The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
- The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
- The water temperature, the oxygen concentrations and pH of the controls and the test material solution were measured at the beginning and at the end of the renewal periods.

RANGE-FINDING STUDY
- A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions.
- Test concentrations: 0.1, 1, 10 and 100 % saturated solutions preparaed by appropriate dilution of the stock solution.
- Results used to determine the conditions for the definitive study: Yes. Because no toxic response was observed during the preliminary concentration rangefinding test, a Limit Test was carried out using only one test concentration at the solubility level of the test material in the test medium (100 % saturated solution) and one control group in a semi-static system. The biological results are based on both of the nominal and measured geometric mean test material concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2.89 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: > 100 % saturated solution

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 2.89 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: > 100 % saturated solution

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.89 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 100 % saturated solution

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

2.89 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 100 % saturated solution

Details on results:

CONCENTRATIONS OF THE TEST MATERIAL
- Test concentration was analytically determined at the beginning and at the end of the renewal periods. Samples from the control were also analysed at the start and at the end of the renewal periods.
- The corresponding measured geometric mean test material concentration was: 2.89 μg/L.
- The biological results are based on both of the nominal and measured geometric mean test material concentrations.

IMMOBILISATION
- The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour (Table 1).
- In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Results with reference substance (positive control):

The 24h EC50: 0.65 mg/L, (95 % confidence limits: 0.61 – 0.70 mg/L)

Reported statistics and error estimates:

Due to that 5 % immobilization was observed at the applied test concentration level and taking into account that a limit test was performed, immobilization in control and in treated group was analysed using 2 Sample t-Test (α = 0.05) by TOXSTAT software to compare means.
The EC50, NOEC, LOEC and EC100 values were determined directly from the raw data.

Table 1: Number and percentage of immobilised animals

Test Group	Number of Treated Animals	Number of Immobilised Animals
		24 Hours		48 Hours
		Number	%	Number	%
Control	20	0	0	0	0
100 % Saturated solution	20	0	0	1	5

Table 2: Calculation of Exposure Concentrations

Nominal concentration	Measured concentrations (µg/L)				Geometric mean (µg/L)
	1^strenewal		2^ndrenewal
	Start	End	Start	End
Control	n.d	n.d	n.d	n.d	-
100 % saturated solution	10.094	0.635	8.268	1.316	2.89

n.d.: Not detected.

Validity

- There were no immobilised animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L.

- All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material had no toxic effect at saturation (2.89 μg/L, measured); the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Executive summary:

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2., EPA OCSPP 850.1010, JMAFF 2-7-2-1 and other Japanese guidelines, under GLP conditions.

Acute toxicity of the test material on Daphnia magna was assessed in an Acute immobilisation test, over an exposure period of 48 hours in a semi-static test system.

Because no toxic response was observed during the preliminary range-finding test, a Limit Test was carried out using only one test concentration at the solubility level of the test material in the test medium (100 % saturated solution) and one control group in the definitive test.

The test concentration was analytically determined at the start and at the end of the renewal periods. The corresponding measured geometric mean test item concentration was: 2.89 μg/L. The biological results are based on both of the nominal and measured geometric mean test material concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentration and for the control.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of the test material were the following:

The 24h and 48h EC50 value: > 100 % saturated solution (nominal loading rate of 100 mg/L; 2.89 μg/L, measured).

The 48h EC100 value: > 100 % saturated solution (nominal loading rate of 100 mg/L; 2.89 μg/L, measured).

The 48h No-Observed Effect Concentration (NOEC): 100 % saturated solution (nominal loading rate of 100 mg/L; 2.89 μg/L, measured).

The 48h Lowest Observed Effect Concentration (LOEC): 100 % saturated solution (nominal loading rate of 100 mg/L; 2.89 μg/L, measured).

Under the conditions of this study, the test material had no toxic effect at saturation (2.89 μg/L, measured); the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Description of key information

Under the conditions of this study, the test material had no toxic effect at saturation (2.89 μg/L, measured); the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Additional information

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2., EPA OCSPP 850.1010, JMAFF 2-7-2-1 and other Japanese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Acute toxicity of the test material on Daphnia magna was assessed in an Acute immobilisation test, over an exposure period of 48 hours in a semi-static test system.

Because no toxic response was observed during the preliminary range-finding test, a Limit Test was carried out using only one test concentration at the solubility level of the test material in the test medium (100 % saturated solution) and one control group in the definitive test.

The test concentration was analytically determined at the start and at the end of the renewal periods. The corresponding measured geometric mean test item concentration was: 2.89 μg/L. The biological results are based on both of the nominal and measured geometric mean test material concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentration and for the control.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of the test material were the following:

The 24h and 48h EC50 value: > 100 % saturated solution (nominal loading rate of 100 mg/L; 2.89 μg/L, measured).

The 48h EC100 value: > 100 % saturated solution (nominal loading rate of 100 mg/L; 2.89 μg/L, measured).

The 48h No-Observed Effect Concentration (NOEC): 100 % saturated solution (nominal loading rate of 100 mg/L; 2.89 μg/L, measured).

The 48h Lowest Observed Effect Concentration (LOEC): 100 % saturated solution (nominal loading rate of 100 mg/L; 2.89 μg/L, measured).

Under the conditions of this study, the test material had no toxic effect at saturation (2.89 μg/L, measured); the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.