Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine

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

Study period:

2018-11-12 to 2018-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All loading levels and the control were analytically verified

Vehicle:

no

Details on test solutions:

Six water accommodated fractions (WAF) were prepared 24 ± 1 prior to the start of the exposure.
For each loading level an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide.
The glass slide with the test item was inserted into a brown glass flask with an appropriate amount of demineralized water.
These dispersions were shaken for at least 24 hours with 20 rpm at room temperature.
Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL).
The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded.
The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item.
Thereafter, the filtration was continued. The next 25 mL were discarded.
During filtration, the filter was always be kept covered.
The solutions were checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Six water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 1.00 -3.16 – 10.0 - 31.6 – 100 - 316 mg/L (factor √10).

Test organisms (species):

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

Details on test organisms:

Details on test organisms
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, CCAP 278/4
- Source (laboratory, culture collection): (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts to 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
- Acclimation period: A four days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): Nutrient medium Z according to LÜTTGE et al. (1994)
Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

38 mg CaCO3/L

Test temperature:

22.8

pH:

7.75 - 9.61

Conductivity:

141 µS/cm

Nominal and measured concentrations:

Six water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 1.00 -3.16 – 10.0 - 31.6 – 100 - 316 mg/L (factor √10).

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density:6442 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 μE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm.

TEST CONCENTRATIONS
Six water accommodated fractions (WAF) were prepared with a nominal loading of the test item of 1.00 -3.16 – 10.0 - 31.6 – 100 - 316 mg/L (factor √10).
The loading levels are based on the results of a preliminary range finding test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate, routine testing

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

135 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

50.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Inhibition of yield

Details on results:

Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-09-25 to 2018-09-28.
ErC50 0.799 (95% confidence interval 0.707 – 1.30)

Reported statistics and error estimates:

The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield.

The following statistical tests were conducted:
- Shapiro-Wilk’s test on normal distribution was calculated with a significance level of 0.01.
- Levene’s test on variance homogeneity was calculated with a significance level of 0.01.
- Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm was done for growth rate with a significance level of 0.05.
- Monotonicity of Concentration/Response was calculated by trend analysis by contrasts (significance level 0.05) for yield.
- Williams Multiple Sequential t-test Procedure was performed for yield with a significance level of 0.05.

TOC Content of the Test Media at the Start and the End of the Exposure

Sampling	0 hours Start of the exposure	72 hours End of the exposure
Nominal test item concentration [mg/L]	TOC[mg C/L]
	Meas. TOC [mg C/L]	Meas. TOC [mg C/L]
316	11.9	9.79
100	3.73	3.79
31.6	< LOQ	< LOQ
10.0	< LOQ	< LOQ
3.16	< LOQ	< LOQ
1.00	2.67	2.41
Control	< LOQ	< LOQ

Validity Criteria

Validity Criterion	Required	This study
		Control
Increase of the cell growth in the control cultures	Exponentially≥16-fold corresponding to a specific growth rate of 0.92 day^-1	267-fold (specific growth rate 1.86 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤35%	22.0%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤7%	1.30%

Validity criteria fulfilled:

yes

Conclusions:

In this study, Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values based on nominal test item loadings: The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 135 (123 – 157) mg/L and 50.5 (37.9 – 66.1) mg/L, respectively. The EL10-values with 95% confidence intervals for inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were 45.7 (34.1 – 59.0) mg/L and < 1.00 mg/L, respectively.

Executive summary:

The toxicity of Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016 C.3. 

The aim of the study was the determination of NOEL, LOEL, EL₁₀, EL₂₀- and EL₅₀-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6442 cells/mL. Six water accommodated fractions (WAF) with the nominal test item loadings 1.00 -3.16 – 10.0 - 31.6 – 100 - 316 mg/L (factor √10) were prepared with demineralized water. For each loading level an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a brown glass flask with an appropriate amount of demineralized water. These dispersions were shaken for at least 24 hours with 20 rpm at room temperature. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC,Macherey-Nagel).The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following water soluble fraction (WSFs) were used in the test. During filtration, the filter was always be kept covered. The solutions were checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. The components of the dilution water were added to the WSFs. Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The concentrations of the test item in all loading levels and the control were analytically verified via analysis of total organic carbon at the start of the exposure (fresh media, 0 hours) and at the end of the exposure (aged media, 72 hours). The TOC values at the beginning of the exposure were in a range of < LOQ to 11.9 mg C/L. At the end of the exposure, the TOC values were in the range of < LOQ to 9.79 mg C/L.

 

All effect values given are based on the nominal test item loadings:

 

Growth Rate Inhibition

 

NOEL: 10.0 mg/L

LOEL: 31.6 mg/L

ErL10:  45.7 (34.1 – 59.0) mg/L

ErL20: 72.3 (61.5 – 80.2) mg/L

ErL50: 135 (123 - 157) mg/L

 

Description of key information

In this study, Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values based on nominal test item loadings: The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 135 (123 – 157) mg/L and 50.5 (37.9 – 66.1) mg/L, respectively. The EL10-values with 95% confidence intervals for inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were 45.7 (34.1 – 59.0) mg/L and < 1.00 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

135 mg/L

Additional information

The toxicity of Octadecanoic acid, reaction products with acetic acid andtetraethylenepentamine to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016 C.3. 

The aim of the study was the determination of NOEL, LOEL, EL₁₀, EL₂₀- and EL₅₀-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6442 cells/mL. Six water accommodated fractions (WAF) with the nominal test item loadings 1.00 -3.16 – 10.0 - 31.6 – 100 - 316 mg/L (factor √10) were prepared with demineralized water. For each loading level an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a brown glass flask with an appropriate amount of demineralized water. These dispersions were shaken for at least 24 hours with 20 rpm at room temperature. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC,Macherey-Nagel).The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following water soluble fraction (WSFs) were used in the test. During filtration, the filter was always be kept covered. The solutions were checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. The components of the dilution water were added to the WSFs. Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The concentrations of the test item in all loading levels and the control were analytically verified via analysis of total organic carbon at the start of the exposure (fresh media, 0 hours) and at the end of the exposure (aged media, 72 hours). The TOC values at the beginning of the exposure were in a range of < LOQ to 11.9 mg C/L. At the end of the exposure, the TOC values were in the range of < LOQ to 9.79 mg C/L.

 

All effect values given are based on the nominal test item loadings:

 

Growth Rate Inhibition

 

NOEL: 10.0 mg/L

LOEL: 31.6 mg/L

ErL10:  45.7 (34.1 – 59.0) mg/L

ErL20: 72.3 (61.5 – 80.2) mg/L

ErL50 : 135 (123 - 157) mg/L