1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane 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:

2018-01-16 to 2018-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

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-{[1-(4-methoxyphenyl)propan-2-yl]amino}ethan-1-one hydrobromide

EC Number:

928-779-0

Cas Number:

1178555-23-1

Molecular formula:

C18-H21-N-O4 x HBr

IUPAC Name:

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

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

TH 1165 Keton I Br
Batch number 600CC
solid/white
99.9% w/w purity
MW=396.3 g/mol
no density given
expiry date: 31 Mar 2020
storage conditions: ambient (5-30 deg C), dark, dry

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:

7.57 – 8.25 (control)
7.25 - 8.22 (test concentrations)

Nominal and measured concentrations:

nominal: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control.
geometric mean measured: 0.218, 0.165, 0.122, 0.0927, 0.127 mg/L and a control
The measured initial concentrations of the test item ranged from 96.5 % to 104 % of nominal. In the aged samples the measured concentrations were between

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: 35.53 × 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.:

0.161 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.:

0.125 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.:

0.122 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.165 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Effect concentrations 5

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:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.115 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

After 72 h at termination of the test a concentration response relation was observed for the
inhibition of growth rate and yield from nominal test item concentrations of 3.05 to 100 mg/L. The
inhibition of growth rate peaked at 90.2 % and the inhibition of yield peaked at 99.2 % at a nominal
test item concentration of 100 mg/L.

The morphology of the algae cells was observed microscopically at test end. The cells were
considered normal for the control and up to and including a nominal test item concentration of
31.3 mg/L. No cells were observed at 100 mg/L nominal test item concentration.

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	0.52	1.32	7.69	35.53
0.954	0.52	1.38	8.27	38.42
3.05	0.52	1.87	9.14	42.83
9.77	0.52	1.74	7.41	28.52
31.3	0.52	0.48	0.80	2.45
100	0.52	0.58	0.64	0.79

Table 2: Percentage inhibition of growth rate

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	-5.2	-2.6	-1.7^1)
3.05	-40.0	-6.5	-4.6^2)
9.77	-29.5	1.2	5.0
31.3	115.6	88.3	64.7 *
100	87.7	92.5	90.2 *

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

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	-7.5	-8.1	-8.3^1)
3.05	-68.8	-20.2	-20.9^2)
9.77	-52.5	3.9	20.0
31.3	105.0	96.1	94.5 *
100	92.5	98.3	99.2 *

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	0.945		99.1	13.3	0.127
	24 h aged	0.330	34.6
	48 h aged	< LOD^1)	3.00
	72 h aged	< LOD^1)	3.00
3.05	0 h fresh	3.16	104		3.04	0.0927
	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	9.55	97.8		1.25	0.122
	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	31.6	101		0.527	0.165
	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	96.5	96.5		0.218	0.218
	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 are as follows:
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.161 mg/L and 0.135 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.125 mg/L and 0.115 mg/L, respectively. The NOEC for both inhibition of growth rate and yield after 72 hours were 0.122 mg/L. All results are based on geometric mean measured concentrations.