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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-04-01 to 2016-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 2006-03-23 (corrected 2011-07-28)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

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 measurement of the actual concentrations of the test item, duplicate 10 mL samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae).
For sampling of the 10 mL samples at the end of the test, the test media of the treatment replicates were pooled.
The concentrations of the test item were determined in one of the duplicate 10 mL samples from all test concentrations and from the control from all sampling dates.
From the concentrations measured at the start and the end of the test period, the mean measured concentrations were calculated. For this, the geometric mean was used, as a decrease of test item concentrations during the test period was observed.

- Sample storage conditions before analysis:
All samples were deep-frozen (at about -20 °C) immediately after sampling and stored until analysis.
In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Pre-experiment solubility (in reconstituted test water according to ISO 6341):
To determine the dissolution of the test item in test water and to determine the optimum time period to reach equilibration within a reasonable time period, 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 solution was reached after a stirring period of 24 hours.

- Preparation of test media (based on the OECD Series No. 23 (2000): test media used for the main test were prepared at the start of the test by mixing 300.1 mg of the test item into 3000 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 the highest test medium and was diluted with test water for the preparation of the test media with lower test concentrations.
The test media was prepared just before start of the test.

CONTROL
Six replicates (test water without test item) were tested in parallel. 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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain No.: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no data

Hardness:

calculated water hardness (AAP Medium): 0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

23 °C

pH:

Control (at start of test): 7.2
Control (at end of test): 7.9
Treatment (at start of test): 7.3
Treatment (at end of test): 8.1 to 8.2

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: The undiluted filtrate of a supersaturated dispersion of the test item in test water with a loading rate of 100 mg/L and the dilutions 1:2.2, 1:4.6, 1:10 and 1:22 of the undiluted filtrate
Geometric mean measured concentrations: 119 µg/L (undiluted filtrate), 51 µg/L (1:2.2), 24 µg/L (1:4.6), 11 µg/L (1:10) and 4.4 µg/L (1:22)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (volume 75 mL), covered with a glass dish
The test flasks were incubated in a temperature controlled orbital shaker. The test flasks were positioned randomly and repositioned daily.
- Test volume: 30 mL
- Initial cells density (nominal): 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted test water (AAP Medium) prepared according to the test guidelines.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- pH: it was measured and recorded in each treatment at the start and end of the test.
- Temperature: temperature in the incubator was monitored and recorded continuously.
- Photoperiod: continues illumination by LED light
- Light intensity: approx. 77 μE s^-1 m^-2 (range: 75 to 79 μE s^-1 m^-2)
The light intensity over the incubation area was within a ± 15 %-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: algal biomass was determined daily by fluorescence measurement, which was performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.
- At the end of the test, a sample was taken from the control and from the highest test concentration (undiluted filtrate) to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected.

RANGE FINDING STUDY
Based on these results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item in the test water the following nominal concentrations of the test item were selected for the main test: The undiluted filtrate of a supersaturated dispersion of the test item in test water with a loading rate of 100 mg/L and the dilutions 1:2.2, 1:4.6, 1:10 and 1:22 of the undiluted filtrate.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 119 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 119 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 119 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

17 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

other: yield

Details on results:

Exponential growth in the control (for algal test): yes

CONCENTRATIONS:
During the test period of 72 hours, the test item concentrations in the test media decreased and at test end they were in the range from 75 to 65 % of the initially measured values. Thus, geometric mean measured concentrations were applied to calculate respective ECs.

MICROSCOPIC EXAMINATION:
At the end of the test there were no differences between the algae growing at the test concentration of 119 μg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this test concentration.

TEST MEDIA:
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

PHYSIOLOGICAL DATA:
The environmental conditions (pH-value, room temperature) were determined to be within the acceptable limits.

Results with reference substance (positive control):

Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The following results were obtained by the
laboratory (in life phase, December 2015): EC50 (growth rate, 72 hours): 0.74 mg/L
The EC50 showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.60-1.03 mg/L).

Reported statistics and error estimates:

Inhibition of algal growth was determined from the following growth parameters: specific growth rate and yield.
Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment.
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far aspossible by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by the Williams t-test.
Statistical analysis was performed using ToxRat Professional®.

Table 1: Biomass of Algae

Mean measured test item concentration [µg/L]	Rep. no.	Biomass of Algae*
		24 Hours	48 Hours	72 Hours
Control	1	5.8	43.8	215.4
	2	5.9	41.1	221.2
	3	5.5	39.1	214.5
	4	6.1	43.1	229.2
	5	6.1	39.5	209.7
	6	4.7	38.9	215.6
	Mean	5.7	40.9	217.6
	SD	0.5	2.1	6.7
4.4	1	5.8	43.0	211.9
	2	4.7	39.6	223.6
	3	4.8	40.8	205.1
	Mean	5.1	41.2	213.5
	SD	0.6	1.7	9.3
11	1	5.7	41.1	198.2
	2	5.2	41.4	209.9
	3	5.2	39.3	206.8
	Mean	5.3	40.6	205.0
	SD	0.3	1.2	6.0
24	1	5.8	36.3	186.0
	2	5.0	36.2	180.0
	3	4.5	33.5	162.4
	Mean	5.1	35.3	176.1
	SD	0.6	1.6	12.3
51	1	5.7	35.3	175.9
	2	4.7	35.7	189.1
	3	4.3	33.7	174.0
	Mean	4.9	34.9	179.7
	SD	0.7	1.1	8.2
119°	1	3.7	25.2	120.9
	2	3.8	24.2	129.3
	3	4.5	24.8	128.8
	Mean	4.0	24.7	126.3
	SD	0.4	0.5	4.7

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

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 10⁴). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.835 x 10⁴relative fluorescence units.

Table2: Average growth rates (µ)

Mean measured test item concentration [µg/L]	Average growth rate µ(day^-1) and inhibition of µ(Ir)
	0-24 h		0-48 h		0-72 h
	µ	Ir[%]	µ	Ir[%]	µ	Ir [%]
Control	1.915	0	1.946	0	1.854	0
4.4	1.800	6	1.949	-0.2	1.848	0.3
11	1.854	3.2	1.942	0.2	1.834(*)	1.1
24	1.810	5.5	1.872*	3.8	1.783*	3.8
51	1.760*	8.1	1.867*	4.1	1.790*	3.4
119°	1.560*	18.5	1.694*	12.9	1.673*	9.8

°:          Undilutedfiltrateofa suspensionwitha loading rate of 100mg/L.

*:            Meanvaluestatisticallysignificantlylower than in the control(accordingto aWilliamst-testone-sided smaller,α=0.05).

(*):          Statisticallysignificantly differentfrom the controldueto verylowvariabilityofresults,however not estimatedas a biologicallyrelevanttoxiceffect, sincethe inhibitionwas< 10 %.

Table 3: Yield(Y)

Mean measured test item concentration [µg/L]	Yield Y (x10^4) and Inhibitionof Y(Iy)
	0-24 h		0-48 h		0-72 h
	Y	Iy[%]	Y	Iy[%]	Y	Iy[%]
Control	4.9	0	40.1	0	216.8	0
4.4	4.2	12.7	40.3	-0.6	212.7	1.9
11	4.5	7.3	39.7	0.8	204.1(*)	5.8
24	4.3	11.5	34.5*	13.9	175.3*	19.1
51	4.1*	16.5	34.1*	15.0	178.8*	17.5
119°	3.2*	35.0	23.9*	40.4	125.5*	42.1

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

*:  Mean value statistically significantly lower than in the control (according to aWilliamst-test one-sidedsmaller,α=0.05).

(*): Statistically significantly different from the control due to very low variability of results, however not estimated as a biologically relevant toxic effect, since the inhibition was< 10 %

Table 4: Section-by-sectiongrowth rates

Meanmeasuredtestitemconcentration [µg/L]	Section-by-section growth rates(day^-1)and inhibition of the growth rates(Ir)
	0-24 h		24-48 h		48-72 h
	µ	Ir [%]	µ	Ir[%]	µ	Ir[%]
Control	1.915	0	1.976	0	1.672	0
4.4	1.800	6	2.097	-6.1	1.646	1.5
11	1.854	3.2	2.030	-2.7	1.62	3.1
24	1.810	5.5	1.934	2.1	1.606	4.0
51	1.760	8.1	1.973	0.1	1.638	2.0
119°	1.560	18.5	1.828	7.5	1.631	2.4

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

Table 5: Results for Test Samples

Sampling Day       [d]	Loading Rate 100 mg/L	Measured Concentration of Test Item   x[mg/L]	Sample Preparation Factor   F	Determined Concentration of Test Item   c[mg/L]	% of the Initially Measured Concentration   [%]
0	Control	n.d.	1.25	<LOQ	n.a.
(fresh)	Dilution1:22	0.00432	1.25	0.00540	n.a.
	Dilution1:10	0.00977	1.25	0.0122	n.a.
	Dilution1:4.6	0.0225	1.25	0.0281	n.a.
	Dilution1:2.2	0.0480	1.25	0.0600	n.a.
	UndilutedFiltrate	0.110	1.25	0.138	n.a.
3	Control	n.d.	1.25	<LOQ	n.a.
(aged)	Dilution1:22	0.00283	1.25	0.00353	65
	Dilution1:10	0.00729	1.25	0.00911	75
	Dilution1:4.6	0.0159	1.25	0.0199	71
	Dilution1:2.2	0.0349	1.25	0.0437	73
	UndilutedFiltrate	0.0821	1.25	0.103	74

 LOQ           =0.00254 mg test item/L

n.d.             =not detected

n.a.             =not applicable

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the substance on the growth of the freshwater green algae Pseudokirchneriella subcapitata was investigated in a 72-hour static test under GLP according to OECD 201.
An undiluted filtrate with a loading rate of 100 mg/L was used as the highest test medium and was diluted with test water for the preparation of the test media with lower test concentrations: 1:2.2, 1:4.6, 1:10 and 1:22.
The mean measured test concentrations during the test period were calculated as geometric mean of the measurements at the start and end of the test. During the test period of 72 hours, the test item concentrations in the test media decreased and at test end they were in the range from 75 to 65% of the initially measured values.
The respective EC10 and EC50 for the inhibition of growth rate are above the highest nominal loading rate of 100 mg/L test substance, corresponding to a geometric mean measured concentration of 119 ug/L test substance.

Description of key information

ErC10 (72 h) > 119 µg/L
ErC50 (72 h) > 119 µg/L
(Effect values are based on geometric mean measured concentrations of the dissolved test substance.)

Key value for chemical safety assessment

EC50 for freshwater algae:

119 µg/L

EC10 or NOEC for freshwater algae:

119 µg/L

Additional information

The toxicity of the substance on the growth of the freshwater green algae Pseudokirchneriella subcapitata was investigated in a 72-hour static test under GLP according to OECD 201.

An undiluted filtrate with a loading rate of 100 mg/L was used as the highest test medium and was diluted with test water for the preparation of the test media with lower test concentrations: 1:2.2, 1:4.6, 1:10 and 1:22.

The respective EC10 and EC50 for the inhibition of growth rate are above the highest nominal loading rate of 100 mg/L test substance, corresponding to a geometric mean measured concentration of 119 µg/L test substance.