2-Propenamide, 3-(1,3-benzodioxol-5-yl)-N,N-diphenyl-, (2E)-

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:

2016-03-17 to 2016-04-28

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:

adopted 2004-04-13

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-12-16

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

yes

Details on sampling:

- Sampling method: for the determination of the actual test item concentration, duplicate samples were taken at the start and end of each test medium renewal periods from the test concentration and from the control.
For sampling from the aged test medium, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: all samples were stored deep-frozen (at about -20 °C) immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Pre-experiment solubility in test medium according to ISO 6341:
To determine the dissolution of the test item in test water and to determine the optimum time period to reach steady state, a stirring pre-experiment was performed. Three individual suspensions of the test item with a loading rate of 100 mg/L were stirred for 3, 24 and 96 hours. After stirring, the suspensions were filtered and the concentrations were analytically determined:
3 hours: 6.5 μg/L
24 hours: 125 μg/L
96 hours: 114 μg/L
The maximum concentration of dissolved test item in test water was reached after a stirring period of 24 hours.

- Preparation of test media (based on the OECD Series No. 23 (2000)): test medium used for the main test were prepared at the start of the two test medum renewal periods by mixing nominal 200 mg (effective range: 200.0 and 200.1 mg) of the test item into 2000 mL of test water. 15 minutes ultrasonic treatment and stirring for 24 hours in the dark was applied based on the results of the stirring experiment. Then, the suspension was filtered through a filter, which was pre-conditioned with 1000 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material.
The undiluted filtrate with a loading rate of 100 mg/L was used as test medium.
The test medium was prepared just before the introduction of the daphnids and prior to the test medium renewal after 24 hours.

- Controls: control was run in parallel (test water without addition of the test item). The test water used for the control was treated in the same way as the test media (i.e. 24 hour stirring followed by filtration).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna Straus
- Origin: Daphnia Collection of the University of Basel/Switzerland
- Breeder: IES Ltd Laboratories / Witterswil / Switzerland
- Age at study initiation: 6 - 24 hours old
- Method of breeding/feeding: daphnids are bred under temperature and light conditions identical to those of the tests.
Daphnids were fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG and cultivated under standardized conditions or a mixture of this algal suspension and a commercial fish diet.
- Feeding during test: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

no data

Hardness:

2.5 mmol (250 mg/L as CaCO3)

Test temperature:

21 °C

pH:

0 h: 7.8 (control and test item)
24 h: 7.8 (control and test item)
48 h: 7.9 (control and test item)

Dissolved oxygen:

0 h: 8.5 mg/L (control) and 8.3 mg/L (test item)
24 h: 8.6 mg/L (control and test item)
48 h: 8.7 mg/L (control) and 8.6 mg/L (test item)

Salinity:

not applicable

Nominal and measured concentrations:

nominal loading of the test material: 100 mg/L
mean measured test item concentration: 79 µg/L
For each test medium renewal period, the mean concentration was calculated as geometric mean of the test item concentrations measured at the start (freshly prepared medium) and the end (aged medium) of the test medium renewal period. From the values obtained, the mean measured test item concentration during the test period was calculated as arithmetic mean.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (volume: 100 mL) covered with transparent disposable polypropylene sheets to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Test volume per vessel: 50 mL
- Aeration: test vessels were not aerated during the test.
- Renewal rate of test solution (frequency/flow rate): test medium renewal after 24 hours was performed.
After 24 hours, the test organisms were transferred by wide-bore pipette in new test vessels with freshly prepared test medium of the corresponding concentration.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Reconstitued test water according to ISO 6341 was used. The test water was aerated prior to the start of the study until oxygen saturation was reached.
- Ca/Mg ratio: 4:1 ratio
- Na:K ratio: 10:1 ratio
- The appearance of the test medium was visually controlled.

OTHER TEST CONDITIONS
- Photoperiod: 16 h/8 h light/dark cycle with a 30 minute transition period
- Light intensity: approx.between 15 and 18 μmol m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Biological parameters: immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure.

RANGE FINDING STUDY
The limit test was based on the results of a range-finding test where no toxic effect was observed on the daphnids up to the loading rate of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 79 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

79 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Details on results:

CONCENTRATIONS:
- measured concentration of the test item in the test medium with a loading rate of 100 mg/L was 93 and 72 μg/L at the start of the renewal periods.
- at the end of the two 24-hour renewal periods, 89 and 94% of the initially measured concentration were found. Thus, the test item was sufficiently stable during the test medium renewal periods of 24 hours.

BIOLOGICAL DATA
No immobilized test organisms were determined in the control and at the limit test concentration of 79 μg/L (loading rate of 100 mg/L).

TEST MEDIUM
No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solutions throughout the entire test duration.

Results with reference substance (positive control):

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year and the following results were obtained by the laboratory (December 2015):
EC50 (24 h): 0.78 mg/L
This showed that the sensitivity of the test organisms was within the range given by the guideline (EC50 (24 h): 0.60-2.1 mg/L).

Reported statistics and error estimates:

The 48-hour EL-value could not be calculated due to the absence of a toxic effect of the test item. Therefore, the EC50/EL50 and the NOEC/NOEL were
directly determined from the raw data.
Since an immobilisation of daphnia magna was not observed at the tested limit concentration, the EC50 (48 h) was estimated with > limit concentration.

Table 1: Effect of the test item on the mobility of Daphnia magna

Mean measured test item concentration	No. of daphnids tested	Immobilized daphnids after 24 hours		Immobilized daphnids after 48 hours
[µg/L]		No.	[%]	No.	[%]
Control	20	0	0	0	0
79*	20	0	0	0	0

*: Undiluted filtrate of a suspension with a loading rate of 100 mg/L.

Table 2: Results for test samples

Sampling day/ sample age	Loading rate of 100 mg test item/L	Measured concentration of test item x			Sample preparation factor F	Determined average concentration of test item c	% of the initially measured concentration
		Sample 1	Sample 2	Average
[day/hours]	[mg/L]	[mg/L]	[mg/L]	[mg/L]		[mg/L]	[%]
0/0	Control	n.d.	n.d.	n.a.	1.25	<LOQ	n.a.
	Undiluted Filtrate	0.0752	0.0743	0.0747	1.25	0.0934	n.a.
1/24	Control	n.d.	n.d.	n.a.	1.25	<LOQ	n.a.
	Undiluted Filtrate	0.0672	0.0663	0.0667	1.25	0.0834	89
1/0	Control	n.d.	n.d.	n.a.	1.25	<LOQ	n.a.
	Undiluted Filtrate	0.0586	0.0573	0.0580	1.25	0.0724	n.a.
2/48	Control	n.d.	n.d.	n.a.	1.25	<LOQ	n.a.
	Undiluted Filtrate	0.0547	0.0547	0.0547	1.25	0.0684	94

LOQ    = 0.00254 mg test item /L

n.d.     = not detected

n.a.     = not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

 

Validity criteria fulfilled:

yes

Conclusions:

The undiluted filtrate of the test substance had no acute toxic effects on Daphnia magna in a 48-hour semi-static test up to the analytically measured concentration of 79 µg/L which corresponds to the solubility limit of the test item in test water at a loading rate of 100 mg/L.

Description of key information

EC50 (48 h) > 79 µg/L
(Effect value based on arithmetic mean measured concentration of the dissolved test substance)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 79 µg/L

Additional information

The acute toxicity of the test substance to Daphnia magna was determined in a 48-hour semi-static test under GLP according to the OECD Guideline 202. A limit test was performed to demonstrate that the test item has no toxic effect on the test organisms up to and including the loading rate of 100 mg/L. The undiluted filtrate was applied; the measured concentration of the test substance in the test medium with a loading rate of 100 mg/L was 93 and 72 µg/L at the start of the renewal periods. At the end of the two 24-hour renewal periods, 89 and 94% of the initially measured concentration were found. The test item had no acute toxic effects on Daphnia magna up to the analytically measured concentration of 79 µg/L which corresponds to the solubility limit of the test item in test water at a loading rate of 100 mg/L (respective 48-h EC50 for immobilization > 79 ug/L test substance).