1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with C18-unsatd. fatty acids, Me sulfates (salts)

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Physical state: liquid
- Analytical purity: 100%

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with a loading rate of 10 mg/L applying 2 hours of magnetic stirring to reach maximum solubility of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 10 mg/L concentration in test medium. All test vessels were pre-incubated for 30 minutes with the respective test solution before the start of the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata, NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture.
- Age of inoculum (at test initiation): 4 d (final test), 3 d (range-finding test)
- Method of cultivation: stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C

ACCLIMATION
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.5 - 22.5°C

pH:

8.1-8.3

Nominal and measured concentrations:

nominal: 0, 0.10, 0.32, 1.0, 3.2 and 10 mg/L + 4 mg humic acid per litre
measured (TWA): 0.042, 0.14, 0.39, 1.3 and 1.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Initial cells density: 1E04 cells/mL
- Control end cells density: 66.1E04 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: M2; according to the OECD 201 Guideline, formulated using Milli-RO water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: TLD-lamps type ‘Cool-white’ 30 Watt, light intensity 94 to 107 E/m²/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
- Other: At the end of the test microscopic observations were performed on the lowest test concentration (range-finding test) or on the test concentration closest to the EC50 (both final tests) and 1 lower concentration (final test with humic acid) to observe for any abnormal appearance of the algae

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 (sqrt(10))
- Range finding study
- Test concentrations: nominal 0, 0.010, 0.10, 1.0 and 10 mg/L, without humic acid
- Results used to determine the conditions for the definitive study: ECr50 between nominal concentrations of 0.10 and 1.0 mg/L, EyC50 between nominal concentrations of 0.010 and 0.10 mg/L (without humic acid)
At the start of the test, the actual test concentrations were in agreement with nominal, i.e. 86-87% of initial. These concentrations decreased to 2-39% of initial after 24 hours of exposure and further to 1-13% of initial et the end of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.39 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.54 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% ci 0.10-2.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% ci 0.24-6.0 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.39 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.49 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% ci 0.22-1.0 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.95 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% ci 0.44-2.1 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations revealed no abnormalities

After 48 hours of exposure a brown layer was observed sticking to the glass just above the medium surface at an average concentration of 1.7 mg/L. After 72 hours of exposure a light floating layer and precipitate was observed at an average concentration of 0.39 mg/L; a floating layer, precipitate and a brown layer was observed sticking to the glass just above the medium surface at average exposure concentrations of 1.3 and 1.7 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50 = 0.97 mg/L, 95% confidence interval 0.62 to 1.5 mg/L; historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-Test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.

At the start of the test, the actual test concentrations were in agreement with nominal, i.e. 107-115% of initial. These concentrations decreased to 15-57% of initial after 24 hours of exposure and further to 1-6% of initial et the end of the test. The average exposure concentrations were calculated to be: 0.042, 0.14, 0.39, 1.3 and 1.7 mg/L.

At the start of the test measured T(D)OC concentrations ranged between 1.7 and 2.3 mg/L for all concentrations up to and including 1.0 mg/L. These concentrations remained stable during the test. Measured T(D)OC at 3.2 and 10 mg/L were 3.2 and 6.4 mg/L, respectively, at the start of the test and decreased to 2.5 and 1.6 mg/L, respectively, at the end of the test.

Mean cell densities (x 1E04 cells/mL) during the final test with humic acid

Average conc.	Exposure time (hours)
(mg/L)	0	24	48	72
Humic acis-control	1.0	2.5	14.6	66.1
0.042^1	1.0	2.6	12.9	73.7
0.14^1	1.0	2.0	11.9	72.6
0.39^1	1.0	3.1	11.1	66.2
1.3^2	1.0	2.4	6.6	33.4
1.7^3	1.0	1.0	1.0	1.0

¹Clear yellow solution

²Slightly hazy yellow solution

³Hazy yellow solution

 

 

Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test with humic acid

Average conc.	Mean growth rate		Yield (0-72 h)
(mg/L)	µ (0-72 h)	Reduction (%)	x10^4cells/mL	Inhibition (%)
Humic acid-control	0.05815		65.13
0.042	0.05971	-2.7	72.67	-11.6
0.14	0.05951	-2.3	71.58	-9.9
0.39	0.05809	0.1	65.17	-0.1
1.3	0.04870	16.3	32.42	50.2
1.7	0.00000	100.0	0.00	100.0

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

The NOEC, EC10 and EC50 of MDIPA Esterquat C18 unsatd. to algae in the presence of humic acid based on growth rate were 0.39, 0.54 (95% c.i. 0.10-2.9 mg/L) and 1.2 mg/L (95% c.i. 0.24-6.0 mg/L), respectively.
The NOEC, EC10 and EC50 based on based on yield were 0.39, 0.49 (95% c.i. 0.22-1.0 mg/L) and 0.95 mg/L (95% c.i. 0.44-2.1 mg/L), respectively.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata, strain NIVA CHL 1 were exposed to MDIPA Esterquat C18 unsatd. at nominal concentrations of 0 (control), 0.10, 0.32, 1.0, 3.2 and 10 mg/L, corresponding to measured (TWA) 0.042, 0.14, 0.39, 1.3 and 1.7 mg/L in the presence of 4 mg/L humic acid under static conditions in accordance with OECD guideline 201 (adopted March 23, 2006; Annex 5 corrected 28 July 2011).

Microscopic observations revealed no abnormalities.

The NOEC, EC10 and EC50 based on growth rate were 0.39, 0.54 (95% c.i. 0.10-2.9 mg/L) and 1.2 mg/L (95% c.i. 0.24-6.0 mg/L), respectively.

The NOEC, EC10 and EC50 based on based on yield were 0.39, 0.49 (95% c.i. 0.22-1.0 mg/L) and 0.95 mg/L (95% c.i. 0.44-2.1 mg/L), respectively.

All effect levels are given based onmeasured (TWA) concentrations.

 

 

Results Synopsis

 

Test Organism:Pseudokirchneriella subcapitata, strain NIVA CHL 1

Test Type: Static

 

72 hr ErC10: 0.54 mg a.i./L                  95% c.i. 0.10-2.9 mg/L

72 hr ErC50: 1.2 mg a.i./L                    95% c.i. 0.24-6.0 mg/L

72 hr EyC10: 0.49 mg a.i./L                  95% c.i. 0.22-1.0 mg/L

72 hr EyC50: 0.95 mg a.i./L                 95% c.i. 0.44-2.1 mg/L

72 hr NOEC: 0.39 mg a.i./L

Endpoint(s) Effected:  growth rate, yield

Description of key information

72 h NOEC (growth rate) = 0.39 mg/mL, 72 h ErC10 = 0.54 mg/L (95% c.i. 0.10-2.9 mg/L), 72 h ErC50 = 1.2 mg/L (95% c.i. 0.24-6.0 mg/L);
72 h NOEC (biomass) = 0.39 mg a.i./mL, 72 h EyC10 = 0.49 mg/L (95% c.i. 0.22-1.0 mg/L), 72 h EyC50 = 0.95 mg/L (95% c.i. 0.44-2.1 mg/L)
(Pseudokirchneriella subcapitata); OECD guideline 201, static, GLP, RL1; in the presence of 4 mg/L humic acid 

Key value for chemical safety assessment

EC50 for freshwater algae:

1.2 mg/L

EC10 or NOEC for freshwater algae:

0.54 mg/L

Additional information

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata, strain NIVA CHL 1 were exposed to MDIPA Esterquat C18 unsatd. at nominal concentrations of 0 (control), 0.10, 0.32, 1.0, 3.2 and 10 mg/L, corresponding to measured (TWA) concentrations of 0.042, 0.14, 0.39, 1.3 and 1.7 mg/L in the presence of 4 mg/L humic acid under static conditions in accordance with OECD guideline 201 (adopted March 23, 2006; Annex 5 corrected 28 July 2011).

Microscopic observations revealed no abnormalities.

The NOEC, EC10 and EC50 based on growth rate were 0.39, 0.54 (95% c.i. 0.10-2.9 mg/L) and 1.2 mg/L (95% c.i. 0.24-6.0 mg/L), respectively.

The NOEC, EC10 and EC50 based on based on yield were 0.39, 0.49 (95% c.i. 0.22-1.0 mg/L) and 0.95 mg/L (95% c.i. 0.44-2.1 mg/L), respectively.

All effect levels are given based on measured (TWA) concentrations.

 

Similar results were obtained with the closely related read-across substances MDEA-Esterquat C16-18 and C18 unsatd. and MDEA-Esterquat C16-18 and C18 unsatd.:

As a weight of evidence data are available from a substance with the same structure as MDEA-Esterquat C16-18 and C18 unsatd., only the distribution of fatty acid moiety is shifted to higher amount of unsaturated C18' (Iodine value 47 instead of 25). From a higher amount of unsaturated C18 a higher toxicity is to be expected. In this 72 -h algal study conducted according to OECD TG 201, Scenedesmus subspicatus (new name Desmodesmus subspicatus) were exposed to the similar substance ethanaminium, 2-hydroxy-N-(-2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18 unsatd. fatty acids, chlorides (Iodine value of 47 instead of 25, the highest value for the submitted substance) at measured concentrations of 0, 1.5, 2.8, 6.6, 16, 29 and 64 mg/L under static conditions. The 72 -h EC50 based on biomass is 3.2 mg/L with 95% confidence limits of 2.7 - 3.6 and the 72 -h EC50 based on growth rate is 6.3 mg/L with 95% confidence limits of <1.5 - >64. The 72 -hr NOEC based on growth rate and biomass is 1.5 mg/L (measured concentrations each).

 

In a 96 h algal toxicity study conducted according to a modification of EPA OTS 797.1050 (Algal Toxicity, Tiers I and II), Selenastrum capricornutum was exposed MDEA-Esterquat C16-18 and C18 unsatd. at nominal concentrations of 0, 2, 4, 8, 16 and 32 mg/L under static conditions. The 96 hr ErC50 value was 2.9 mg/L ( 95% confidence limit <2.0 - 4.2 mg/L; nominal).

This toxicity study is classified as not reliable and does not satisfy the guideline requirements for aquatic toxicity to algae (validity Klimisch 3). Deviations were: no analytical determination of test substance, river water used instead of algal media, and cell counts only done on controls at 0 h timepoint.

 

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata, strain NIVA CHL 1 were exposed to MDIPA-Esterquat C16-18 and C18 unsatd. at nominal concentrations of 0 (control), 0.25, 0.80, 2.5, 8.0 and 25 mg/L, corresponding to measured (TWA) 0.29, 0.91, 2.7, 6.2 and 25 mg/L in the presence of 4 mg/L humic (<2 mg/L DOC) acid under static conditions in accordance with OECD guideline 201 (adopted March 23, 2006; Annex 5 corrected 28 July 2011). Microscopic observations revealed no abnormalities. The NOEC, EC10 and EC50 based on growth rate were 2.7, 3.2 (95% c.i. 2.5-4.1 mg/L) and 8.1 mg/L (95% c.i. 6.4-10 mg/L), respectively. All effect levels are given based on measured (TWA) concentrations.