N,N'-[1,4-phenylenebis(methylene)]bis{N,N-dimethyl-3-[(prop-2-enoyl)amino]propan-1-aminium} dichloride

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:

27 Feb - 02 Mar 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

Temperature during test was 20.1–23.6 °C (OECD 201: 21-24 °C ± 2°C), pre-culture preparation: 5 days before test start (OECD 201: 2-4 days before stest start). As exponential growth was observed in the blank control, this was stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland-Pfalz, Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

{[4-({dimethyl[3-(prop-2-enamido)propyl]azaniumyl}methyl)phenyl]methyl}dimethyl[3-(prop-2-enamido)propyl]azanium dichloride

EC Number:

812-548-5

Cas Number:

1621424-03-0

Molecular formula:

C24H40Cl2N4O2

IUPAC Name:

{[4-({dimethyl[3-(prop-2-enamido)propyl]azaniumyl}methyl)phenyl]methyl}dimethyl[3-(prop-2-enamido)propyl]azanium dichloride

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations (0 h, 72 h)

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 220.2 mg/L in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution of this stock solution with algal medium.
- Controls: algal medium only

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: 86.81
- Source: obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of University Göttingen, Germany).
- Age of inoculum (at test initiation): Five days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours.
- Method of cultivation: kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

20.1 - 23.6°C

pH:

7.3 - 7.7

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 10, 22, 46, 100 and 220 mg/L
Measured concentrations initial (t-=0) /test end (t-72h):

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks, total volume 65 mL
- Fill volume: 45 ± 1 mL
- Initial cells density: 2.3*10^3 cells/mL
- Control end cells density: 163567
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium according to OECD 201

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: pH (0, 72 h)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED: cell concentration (0h (control), 24, 48, 72 h)
- Determination of cell concentrations: measured with an electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.09 - 2.2

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

381.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: extrapolated

Remarks:

95% confidence limit: 294.04-489.75 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

105.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limt: 90.36-122.77 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): normal and healthy appearance of algae in the control and all test item concentrations

Reported statistics and error estimates:

The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1.

Any other information on results incl. tables

Table 1: Cell number/mL

 

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2320	9177	37083	163567
Blank control	SD	0	877	2561	9584
10	Mean	2320	8647	34533	156867
10	SD	0	631	3445	11485
22	Mean	2320	8747	34600	166173
22	SD	0	706	461	3900
46	Mean	2320	8813	29793	131633
46	SD	0	431	2281	16586
100	Mean	2320	8600	25680	104540
100	SD	0	788	1482	6702
220	Mean	2320	7807	14567	39073
220	SD	0	130	791	2689

SD = Standard Deviation

Table 2: Daily growth rates of the blank controls

 

Growth rates	days 0 – 1	days 1 – 2	days 2 – 3	CV of sectional daily growth rates	days 0 – 3
Replicate 1	1.380	1.293	1.637	12%	1.437
Replicate 2	1.263	1.429	1.478	8%	1.390
Replicate 3	1.284	1.499	1.485	8%	1.423
Replicate 4	1.401	1.429	1.478	3%	1.436
Replicate 5	1.375	1.459	1.442	3%	1.426
Replicate 6	1.525	1.279	1.388	9%	1.397
Mean	1.371	1.398	1.485	7%	1.418
Standard deviation	0.094	0.090	0.083		0.020
CV	7%	6%	6%		1%

CV = Coefficient of Variation

Table 3: Measured concentrations and % of nominal concentrations

 

Nominal Concentration Test Item	MeasuredConcentrationt = 0h	Measured Concentration t = 72 h	% of Nominal concentration t = 0 h	% of Nominal concentration t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	< LOQ	< LOQ	--	--
10	12.32	12.50	123.20	125.01
22	25.35	26.23	115.23	119.21
46	50.88	53.11	110.60	115.45
100	110.79	116.39	110.79	116.39
220	239.52	252.08	108.87	114.58

LOQ (Limit of quantification) = 4.00 mg/L

* The recovery of the daily prepared QC sample of 95.8% was taken intoaccount.

Table 4: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	71	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	7%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1%	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.