1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jan 2018 - 04 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

EC Number:

222-148-7

EC Name:

1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

Cas Number:

3371-33-3

Molecular formula:

C17H19NO4.BrH

IUPAC Name:

1-(3,5-dihydroxyphenyl)-2-{[1-(4-hydroxyphenyl)propan-2-yl]amino}ethan-1-one hydrobromide

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken and analysed from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution of 100mg/L was prepared. Lower test solutions were prepared by dilution of the stock solution with test medium. Algae were added to each solution individually targeting nominal cell densities of 0.5 × 104 cells per mL in each solution.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae)
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Göttingen
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory.

ACCLIMATION
- Acclimation period: 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Culturing media and conditions (same as test or not): yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.6 – 23.2 °C

pH:

7.49 – 8.30 (control)
7.26 - 8.34 (test concentrations)

Nominal and measured concentrations:

nominal: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control
geometric mean measured: 0.208, 0.158, 0.119, 0.0906, 0.0677mg/L and a control
The measured initial concentrations of analyte ranged from 79.9 % to 94.5 % of nominal. In the aged samples (24, 48 and 72h) the measured concentrations were

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
- Aeration: continuous agitation
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Initial cells density: 0.5 × 10^4 cells/mL.
- Control end cells density: 39.31 × 10^4 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes AAP-Medium ((according to Annex 3 of OECD 201))

OTHER TEST CONDITIONS
- Photoperiod: Continuously
- Light intensity and quality: 88.3 µEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0).)

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.135 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.116 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.119 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.158 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.106 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Reported statistics and error estimates:

Probit analysis following normal distribution for the determination of EC values.
Jonckheere Terpstra test (left-sided, p<0.05) for growth rate and for yield for the determination of NOEC and LOEC.

Any other information on results incl. tables

Table 1: Average cell numbers for each sampling time and concentration

nominal Conc.	Average cell numbers [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.49	1.53	8.05	39.31
0.954	0.49	2.09	10.33	48.66
3.05	0.49	2.11	9.96	42.66
9.77	0.49	1.89	5.57	21.04
31.3	0.49	0.93	0.65	0.70
100	0.49	0.83	0.85	0.92

Table 2: Percentage inhibition of growth rate

nominal Conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.954	-31.5	-9.8	-5.3^1)
3.05	-32.4	-8.4	-2.1^2)
9.77	-22.1	12.6	14.0
31.3	43.3	90.6	92.0 *
100	52.7	80.3	85.7 *^1)

¹⁾Value was omitted for EC_{10, 20, 50}-calculation

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Table 3: Percentage inhibition of yield

niminal Conc.	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.954	-53.8	-30.2	-24.1^1)
3.05	-55.8	-25.3	-8.7^2)
9.77	-34.6	32.8	47.0
31.3	57.7	97.9	99.5 *
100	67.3	95.2	98.9 *^1)

¹⁾Value was omitted for EC_{10, 20, 50}-calculation

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Table 4: Determined concentration of the test item

Test item nominal [mg/L]	Sampling	Test item found				Actual test item conc. [mg/L]
		[mg/L]	% of nominal		Geometric mean [%]
Control	0 h fresh	< LOD	-		-	-
	24 h aged	< LOD	-
	48 h aged	< LOD	-
	72 h aged	< LOD	-
0.954	0 h fresh	0.900		94.3	7.10	0.0677
	24 h aged	< LOD^1)	3.00
	48 h aged	< LOD^1)	3.00
	72 h aged	< LOD^1)	3.00
3.05	0 h fresh	2.88	94.5		2.97	0.0906
	24 h aged	< LOD^1)	0.938
	48 h aged	< LOD^1)	0.938
	72 h aged	< LOD^1)	0.938
9.77	0 h fresh	8.55	87.5		1.22	0.119
	24 h aged	< LOD^1)	0.293
	48 h aged	< LOD^1)	0.293
	72 h aged	< LOD^1)	0.293
31.3	0 h fresh	26.7	85.2		0.505	0.158
	24 h aged	< LOD^1)	0.0914
	48 h aged	< LOD^1)	0.0914
	72 h aged	< LOD^1)	0.0914
100	0 h fresh	79.9	79.9		0.208	0.208
	24 h aged	< LOD^1)	0.0286
	48 h aged	< LOD^1)	0.0286
	72 h aged	< LOD^1)	0.0286

 - = not calculated;LOD = 0.0286 mg/L test item;LOQ = 0.0954 mg/L test item

¹⁾Analyte was below LOD in the sample. Therefore the LOD value was used for calculation of geometric mean according to OECD Guidance document No. 23 (2000)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test substance to the green freshwater algae Pseudokirchneriella subcapitata was determined according to OECD 201 and GLP. The results after 72 hours are as follows:

Significant inhibitory effects were determined for growth rate and yield at test item concentrations
of 31.3 mg/L (nominal) and 0.158 mg/L (actual) and above. The overall LOEC was therefore
determined to be 31.3 mg/L (nominal) and 0.158 mg/L (actual), the corresponding NOEC was set
at 9.77 mg/L (nominal) and 0.119 mg/L (actual).

The EC50-values for inhibition of growth rate (ErC50) was determined to be 16.2 mg/L (nominal) and 0.135 mg/L (actual). And the EC50-value for yield (EyC50) was 10.2 mg/L (nominal) and 0.120 mg/L (actual).

The EC10-values for inhibition of growth rate (ErC10) was determined to be 8.91 mg/L (nominal) and 0.116 mg/L (actual). And the EC10-value for yield (EyC10) was 5.99 mg/L (nominal) and 0.106 mg/L (actual).

The EC20-value for growth rate (ErC20) was determined to be 10.9 mg/L (nominal) and 0.122 mg/L (actual). The EC20-value for yield (EyC20) was 7.18 mg/L (nominal) and 0.111 mg/L (actual).