Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

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:

04 December 2018 - 06 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

EC Number:

291-707-5

EC Name:

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Cas Number:

90459-62-4

Molecular formula:

C24H55N3O6S

IUPAC Name:

bis(2-aminoethyl)amine octadecanoic acid dimethyl sulfate

Test material form:

solid

Remarks:

paste

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations during dose range finding study and main study, day 0(fresh) and day 3(aged)

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Based on the in house dissolution test the test item forms suspension (low soluble in OECD Alga Media), hence OECD Alga Media is selected as a vehicle for preparation of test item formulation.
Individual WAFs were prepared by adding the appropriate amount of test item to dilution water (OECD Alga media) at nominal loading rates of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L for the range finding and 3.5, 6.0, 10.2, 17.3, 29.4 and 50.0 mg/L for the main study. Each mixing vessels were filled to achieve minimal headspace given the constraints of the vessels and closed with foil-covered stoppers.
The mixture was stirred using a magnetic stirrer at 100 RPM for 6 hour (the duration of mixing was 6 hours, selected based on WAF equilibration trial).
Before removing the test solution, the mixtures were allowed to settle and equilibrate to test temperature. The aqueous portions of WAFs were collected using a steri-pipette and transferred to the test vessels. The same method was employed for the control group using OECD alga media.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: ATCC 22662
- Source (laboratory, culture collection): in-house culture
- Age of inoculum (at test initiation): 3 d
- Method of cultivation:
Alga stock culture (maintained in OECD alga medium) is periodically subcultured at least once per week and maintained with the illumination and temperature of 4440 to 8880 lux and 21 to 24°C, respectively. From a healthy axenic culture of alga, a pre-culture of the desired cell density was prepared to provide the inoculum for test cultures.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.2 to 22.7 °C

pH:

main study: 7.67 to 8.07 at the beginning (0 h) and from 7.66 to 8.01 at the end (72 h) of the test with alga medium and from 7.85 to 8.04 at the beginning (0 h) and from 7.79 to 7.96 at the end (72 h) of the test without alga medium

Nominal and measured concentrations:

nominal: 3.5, 6.0, 10.2, 17.3, 29.4 and 50.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilised conical glass flasks (Erlenmeyer flasks) of 250 mL capacity, filled with 100 mL
- Initial cells density: 5775 cells/mL
- Control end cells density: 1003750 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

The cells in the culture flasks were maintained in suspension by agitating the culture medium continuously at 100 revolutions per minute (rpm) using an orbital shaker.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: 5262 to 5876 Lux produced with white fluorescent lamps

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: haemocytometer
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7

- Range finding study
- Test concentrations: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: 100% inhibition of growth rate at 50 and 100 mg/L, 11.31% inhibition at 10 mg/L, 1.21% inhibition at 1 mg/L, 1.34% inhibition at 0.1 mg/L, and 0.59% inhibition at 0.01 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

8.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 7.11 - 10.30 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

25.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 21.93 - 29.88 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

4.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 3.56 - 5.36 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

10 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 9.06 - 11.05 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Any stimulation of growth found in any treatment: no

The alga cells were found healthy and normal in both control and treatment groups and no treatment related changes were observed in the algal cell counts at 24, 48 and 72 hours after exposure during the range finding and the main study.
As the concentration of the test substance after 72 hours has been satisfactorily maintained within 20% of the measured initial concentration throughout the test, the test results are based on nominal values.
There was no evidence of a marked change in the pH of the test medium on day 0 of treatment in the range finding and the main study.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72 hErC50 = 1.11 mg/L, 72 h EyC50 = 0.29 mg/L
This 72 hours growth rate inhibition and percent inhibition in yield lies within the validity criteria acceptance range and establishes the acceptability of test system response and test procedures followed.

Reported statistics and error estimates:

The 72 hours EL10 and EL50 values were calculated from the responses (growth rate and yield) data against log loading rate using the Probit analysis method (Finney, 1971). Probit regression line was drawn for assessed parameters by plotting the probit inhibition values against the corresponding log nominal loading rate.

Any other information on results incl. tables

Validity criteria:

The biomass in the control cultures increased exponentially by a factor of 158.05 and 173.81 within 72 h test period. This corresponds to an average specific growth rate of 1.72 per dayin the range finding and main study respectively (validity criterion: biomass should increase at least by factor of 16 and specific growth rate of 0.92 per day).

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3, for 72-hours test) in the control cultures was 33.79 and 25.81 during the range finding and main study respectively (validity criterion: must not exceed 35%).

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1.69 and 1.72 in the range finding and main study, respectively (validity criterion: should not exceed 7%).

Percent Inhibition of Growth Rate

 

Dose Range Finding Study		Main Study
Nominal Loading Rate (mg/L)	% Inhibition in Growth Rate	Nominal Loading Rate (mg/L)	% Inhibition in Growth Rate
0.0	-	-	-
0.01	0.59	3.5	0.0
0.1	1.34	6.0	6.0
1.0	1.21	10.2	10.1
10.0	11.31	17.3	31.2
50.0	100.0	29.4	59.7
100.0	100.0	50.0	100.0

 

Percent Inhibition in Yield

 

Dose Range Finding Study		Main Study
Nominal Loading Rate (mg/L)	% Inhibition in Yield	Nominal Loading Rate (mg/L)	% Inhibition in Yield
0.0	-	-	-
0.01	3.0	3.5	0.0
0.1	6.7	6.0	26.6
1.0	6.1	10.2	40.9
10.0	43.9	17.3	80.5
50.0	100.0	29.4	95.9
100.0	100.0	50.0	100.0

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

72 h ErL10 = 8.56 mg/L, 95% confidence interval 7.11 - 10.30 mg/L.
72 h EyL10 = 4.37 mg/L, 95% confidence interval 3.56 - 5.36 mg/L.
72 h ErL50 = 25.60 mg/L, 95% confidence interval 21.93 - 29.88 mg/L.
72 h EyL50 = 10.00 mg/L, 95% confidence interval 9.06 - 11.05 mg/L.
72 h NOEL = 3.5 mg/L, LOEL = 6.0 mg/L
All results are based on the nominal loading rate.

Executive summary:

The test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized was assessed on Pseudokirchneriella subcapitata for Growth Inhibition Test as per the OECD Guidelines for Testing of Chemicals (Section 2), Effects on Biotic Systems, Guideline No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” adopted on 23 March 2006.

 

Dose Range Finding Study

A dose range finding study was conducted with six nominal loading rates of 0.01, 0.1, 1.0, 10.0, 50.0 and 100 mg/L along with a control, each loading rate contained 2 replicates except for the control which possessed 3 replicates. The algal growth (cell density) wasassessed during the test at 24, 48 and 72 hours post-exposure.

During the dose range finding study, exponentially growing cells (5742 cells/mL) were exposed to the range of selected nominal loading rates. As compared to the control, the growth rate inhibition of 0.59, 1.34, 1.21, 11.31, 100 and 100% was observed at the tested nominal loading rates of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/Lof test item, respectively.

As compared to the control, an inhibition in yield of 3.0, 6.7, 6.1, 43.9, 100 and 100% was observed at the tested nominal loading rates of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L of test item,respectively.

 

Main Study

Based on the results of dose range finding study, a main study was conducted at the nominal loading rates of 3.5, 6.0, 10.2, 17.3, 29.4 and 50.0 mg/L along with a control. Exponentially growing alga cells (5775 cells/mL) were exposed to the selected nominal loading rates. The algal growth (cell density) was assessed at 24, 48 and 72 hours post-exposure.

As compared to the control, a growth rate inhibition of 0.0, 6.0, 10.1, 31.2, 59.7 and 100 % was observed at the nominal loading rates of 3.5, 6.0, 10.2, 17.3, 29.4 and 50.0 mg/L, respectively.

Similarly, and as compared to the control, an inhibition in yield of 0.0, 26.6, 40.9, 80.5, 95.9 and 100% was observed at the nominal loading rates of 3.5, 6.0, 10.2, 17.3, 29.4 and 50.0 mg/L respectively.

The present alga growth inhibition study fulfills the validity criteria of the test.

Substance concentration during testing was analysed by TOC-Analysis. As the concentration of the test substance has been satisfactorily maintained within 20% of the measured initial concentration throughout the test duration of 72 hours, the results are based on nominal values.

 

Conclusion

Growth Rate (ErL10)

The 72 hours EL10 value for growth rate (ErL10) is determined to be 8.56 mg/L (Effective Loading Rate). The 95% confidence interval value was found to have a lower limit of 7.11 and an upper limit of 10.30 mg/L.

Yield (LyL10)

The 72 hours EL10 value for yield (EyL10) is 4.37 mg/L (Effective Loading Rate). The 95% confidence interval value was found to have a lower limit of 3.56 and an upper limit of 5.36 mg/L.

Growth Rate (ErL50)

The 72 hours EL50 value for growth rate (ErL50) is determined to be 25.60 mg/L (Effective Loading Rate). The 95% confidence interval value was found to have a lower limit of 21.93 and an upper limit of 29.88 mg/L.

Yield (EyL50)

The 72 hours The EL50 value for yield (EyL50) is 10.00 mg/L (Effective Loading Rate). The 95% confidence interval value was found to have a lower limit of 9.06 and an upper limit of 11.05 mg/L.

NOEL and LOEL

The 72 hours No Observed Effect Loading Rate (NOEL) is 3.5 mg/L and Lowest Observed Effect Loading Rate (LOEL) is 6.0 mg/L, respectively.

All results are based on the nominal loading rate.