Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

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:

2018-03-19 to 2018-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

yes

Remarks:

The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

yes

Remarks:

The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

IUPAC Name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

Test material form:

other: solid, very viscous yellow paste

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Nominal test concentrations: 0.032 / 0.1 / 0.32 / 1 / 3.2 / 10 mg/L.
- Sampling method: taken at the start and at the end of the test

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 100.0 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. Lower test concentrations were prepared by dilution of the stock solution in algal medium.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: SAG 86.81
- Source: MBM Sciencebridge GmbH
- Method of cultivation: The algae are 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.
- Pre-Culture: an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.2 – 24.0 °C

pH:

7.6 – 8.0

Nominal and measured concentrations:

Nominal: 0.032 / 0.1 / 0.32 / 1 / 3.2 / 10 mg/L
Measured: 0.029 / 0.091 / 0.292 / 0.911 / 2.917 / 9.115 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Type: open, covered with perforated plastic foil acting as a stopper
- Fill volume: filled with 45 ± 1 mL of the respective test solution
- Initial cells density: 2.4 E+03 cells/mL
- Control end cells density: 497763 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD TG 201 medium

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

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter
- Other: cell number: determined before the start of incubation and every 24 hours; pH value: measured before and after the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125
Range finding study
- Test concentrations: 2 pre-test performed: 1, 10, 100 mg/L and 0.01, 0.1, 1 mg/L
- Results used to determine the conditions for the definitive study: Inhibition for Growth Rate (0-72h): 0.30% for 0.1 mg/L and 120.64% for 10 mg/L

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.23 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.:

0.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Microscopical Observations: No cells visible for 0.32, 1, 3.2, 10 mg/L; Normal and healthy algae observed in blank control, 0.032 and 0.01 mg/L group.
- Analytical determination: The measured concentrations lay between 26 % and 96 % of the nominal concentrations at the beginning of the test and between 0 % and 87 % of the nominal concentrations at the end of the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.90 mg/L for growth rate; 0.42 mg/L for yield

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1.

Any other information on results incl. tables

Non-GLP pre-test:

Experiment	Nominal concentration (mg/L)	% inhibition
		Growth rate (0-72h)	Yield (0-72h)
	control	0	0
Exp. 2	0.01	-4.4	-24.29
	0.1	0.3	1.71
	1	22.06	67.38
Exp. 1	1	78.78	97.48
	10	120.64	101.06
	100	119.82	101.03

Biological results:

Individual cell numbers:

Nominal Conc. in mg/L	Mean cell number/mL
	0 h	24 h	48 h	72 h
Control	2420	12613	87143	497763
0.032	2420	9993	79193	453273
0.1	2420	9433	39473	155047
0.32	2420	9680	17500	21253
1	2420	5993	4687	5993
3.2	2420	4540	3513	2673
10	2420	1413	1247	1513

Analytical results:

Mean measured concentrations and % of nominal:

Nominal Concentration Test Item	Measured concentration  t = 0 h	Measured concentration  t = 72 h	% of nominal  t = 0 h	% of nominal  t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	n.d.	n.d.	--	--
0.032	< LOQ	n.d.	25.76	-5.19
0.1	0.0352	< LOQ	35.24	5.80
0.32	0.2043	0.0633	63.86	19.80
1	0.8317	0.4649	83.17	46.49
3.2	2.8525	2.5823	89.14	80.70
10	9.6171	8.6390	96.17	86.39

Effect levels:

Toxicological endpoints for the test item:

Parameter	Value	95% confidence interval
NOEC (Growth Rate) 72 h	0.03 mg/L	--
NOEC (Yield) 72 h	0.03 mg/L	--
LOEC (Growth Rate) 72 h	0.09 mg/L	--
LOEC (Yield) 72 h	0.09 mg/L	--
72h ErC10	0.05 mg/L	0.03 – 0.07 mg/L
72h EyC10	0.03 mg/L	0.02 – 0.04 mg/L
72h ErC50	0.23 mg/L	0.14 – 0.39 mg/L
72h EyC50	0.07 mg/L	0.05 – 0.09 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72h-EC50 for growth rate and yield were determined to be 0.23 mg/L and 0.07 mg/L, respectively. The 72h-EC10 for growth rate and yield were determined to be 0.05 mg/L and 0.03 mg/L, respectively. The NOEC and LOEC were both 0.03 mg/L and 0.09 mg/L for growth rate and yield.

Executive summary:

The toxicity of test item against Desmodesmus subspicatus was determined according to OECD 201. The study was performed using 5 concentrations ranging from 0.032 to 10 mg/L with a spacing factor of 3.125. Incubation time was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

At test start and end, the content of the test item in the test solutions was determined using LC-MS-MS.The biological results were based on the calculated values (geometric mean).

Significant inhibition of algal growth was observed at the concentrations 0.1 – 10 mg/L. The 72h-EC50 for growth rate and yield were determined to be 0.23 mg/L and 0.07 mg/L, respectively. The 72h-EC10 for growth rate and yield were determined to be 0.05 mg/L and 0.03 mg/L, respectively. The NOEC and LOEC were both 0.03 mg/L and 0.09 mg/L for growth rate and yield.