Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

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:

from 2017-12-11 to 2018-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: For determination of the test item concentrations, three replicate samples (5 mL per replicate) were taken from each test item concentration and from the control. Formulation samples were diluted with mobile phase A and analysed by an HPLC method with MS detection. Control samples were directly injected.
- Sampling method: Samples were taken at the start (0 hours) and at the end of exposure (72 hours)
- Sample storage conditions before analysis: The samples were kept in containers tightly closed in a dry, room temperature and well-ventilated place.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions used in the test was prepared by mechanical dispersion without using any solubilising agents.
For preparation of test solutions a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.078 g test item in 780 mL OECD medium using approx. 10 min shaking to obtain clear solution. The test solutions of subsequent concentrations were prepared by appropriate dilution of this stock solution
- Controls: Untreated control ran parallel in the test. The positive control was performed with potassium dichromate.
- Evidence of undissolved material: No

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: algae
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture was intended to give an amount of algae suitable for the inoculation of test cultures.

ACCLIMATION
- Acclimation period: The pre-culture was incubated for four days at this test.
- Culturing media and conditions: The culture conditions were the same as the test. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing.
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

No data

Test temperature:

The temperature in the flask was in the range of 22.9 – 23.4 °C and within the climate chamber between 22.0 and 23.9 °C.

pH:

The range of the pH was 7.79 – 8.86 during the study.

Dissolved oxygen:

No data

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

- Nominal concentrations: The following nominal concentrations were tested: 1.2, 3.7, 11.1, 33.3 and 100.0 mg/L.
- Measured concentrations: The corresponding calculated geometric mean concentrations were the followings: 0.92, 3.38, 10.77, 33.81 and 103.99 mg/L. Measured concentrations of the components of the test substance ((Phosphoric acid mono(2-ethylhexyl) ester, Pyrophosphoric acid di(2-ethylhexyl) ester and Phosphoric acid bis(2-ethylhexyl) ester)) were in the range of 53 – 119 % of the nominal during the test (i.e. more than ± 20 % deviation) therefore the exposure concentrations were calculated as the geometric mean of the concentrations (based on the mean value of the three components) measured at the start and end of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume
- Fill volume: 100 mL
- Aeration: No
- Initial cells density: The initial cell density was about 10 000 cells/ mL in each test flask.
- Control end cells density: Cell density in the control cultures increased by a factor of more than 16 (by a factor of 61.67) within 72 hours. This corresponds to a specific growth rate of 1.37 day-1.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition, temperature was continuously measured (with a min/max thermometer) within the climate chamber. The pH was checked at the beginning and at the end of the study, in the controls and at each concentration.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The average light intensity measured at the position occupied by algal culture flasks during the test was 7948 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED: The purpose of this study was to determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The algal growth in relation to the untreated control culture was determined over a fixed test period of 72 hours and thus, over several algal generations.
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Morphological Changes of Algal Cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.
- Chlorophyll measurement: Not determined

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: Yes. A non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.
- Test concentrations: Algal cells were exposed to the concentration of 100, 10, 1, 0.1 and 0.01 mg/L test item, plus untreated control, for 72 hours.
- Results used to determine the conditions for the definitive study: In the pre-experiments the 72h-EC50 was determined to be between 10 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

yield

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: Not observed
- Unusual cell shape: Not observed
- Colour differences: Not observed
- Flocculation: Not observed
- Adherence to test vessels: Not observed
- Aggregation of algal cells: Not observed
- Any stimulation of growth found in any treatment: Not observed
- Any observations that might cause a difference between measured and nominal values: Yes. The test item concentrations were calculated based on the measured concentrations of the three components: Phosphoric acid mono(2-ethylhexyl) ester, Pyrophosphoric acid di(2-ethylhexyl) ester and Phosphoric acid bis(2-ethylhexyl) ester. Measured concentrations of these components were in the range of 53 – 119 % of the nominal during the test (i.e. more than ± 20 % deviation) therefore the exposure concentrations were calculated as the geometric mean of the concentrations (based on the mean value of the three components) measured at the start and end of the test.
- Effect concentrations exceeding solubility of substance in test medium: No
- Toxic effects: In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata at the concentrations of 6.7 and 20.0 mg/L. After 72 hours, the Er10 and the ErC50 were determined to be 2.50 mg/L and 9.31 mg/L respectively.The EyC10 and the EyC50 were determined to be 0.82 mg/L and 3.07 mg/L respectively and the 72h-NOEC was determined to be 2.2 mg/L.

Results with reference substance (positive control):

The following results were obtained from the positive control performed with potassium dichromate:
The 72h ErC50: 0.82 mg/L, (95 % confidence limits: 0.57 – 1.25 mg/L)
The 72h EyC50: 0.51 mg/L, (95 % confidence limits: 0.45 – 0.57 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 24 h and 48 h, and at the end of the test (72 hours after the start of the test) using Excel for Windows software.
Percentage inhibition of growth rate (μ) and yield (y) were calculated using Excel for Windows software.
The ECx values and their confidence limits for growth rate and yield were calculated using Probit analysis by SPSS software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett’s test (α = 0.05; 2-sided) by SPSS software.

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata) was determined according to the principles of OECD 201 (2011) under GLP conditions. After 72 hours the ErC10 was determined to be 15.50 mg/L and the ErC50 was determined to be 34.63 mg/L.

Executive summary:

The purpose of this GLP study according to OECD 201 (2011) was to determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) after 72 hours. The study was conducted under static conditions with an initial cell density of 10 000 cells/mL. The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber. The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope. Nominal concentrations of 1.2, 3.7, 11.1, 33.3 and 100.0 mg/L were investigated in the main study. The test solutions used in the test was prepared by mechanical dispersion without using any solubilising agents. An appropriate amount of test item was dissolved in the dilution water (OECD medium) in order to obtain the concentration of 100 mg/L. The further test solutions were prepared by appropriate dilution of this stock solution. After the formulation procedure algal cells were immediately introduced into the test solutions. Untreated control (dilution water (OECD medium) without addition of the test item) ran parallel in the test and a positive control with potassium dichromate was performed. The components of the test item (Phosphoric acid mono(2-ethylhexyl) ester, Pyrophosphoric acid di(2-ethylhexyl) ester and Phosphoric acid bis(2-ethylhexyl) ester) were used for the quantitation of the test item. Samples were taken from each test concentrations and from the control at the start (0 hours) and at the end of exposure (72 hours) and analyzed using a HPLC- MS method. Measured concentrations of these components were in the range of 71 – 103 % of the nominal at the start and 53 – 119 % at the end of the test. The corresponding measured geometric mean test item concentrations were: 0.92, 3.38, 10.77, 33.81 and 103.99 mg/L. Biological results are based on the measured geometric mean test item concentrations as the analytically measured test item concentrations were not within the range of ± 20 % of the nominal over the test period of 72 hours. The 24h-ErC50 and the EyC50 of potassium dichromate was determined to be 0.82 and 0.51 mg/L respectively. The validity criteria were fulfilled. After 72 hours the ErC10 was determined to be 15.50 mg/L and the ErC50 was determined to be 34.63 mg/L.

Description of key information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata) was determined according to the principles of OECD 201 (2011) under GLP conditions. After 72 hours the ErC10 was determined to be 15.50 mg/L and the ErC50 was determined to be 34.63 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

34.63 mg/L

EC10 or NOEC for freshwater algae:

15.5 mg/L

Additional information

The purpose of this GLP study according to OECD 201 (2011) was to determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) after 72 hours. The study was conducted under static conditions with an initial cell density of 10 000 cells/mL. The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber. The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope. Nominal concentrations of 1.2, 3.7, 11.1, 33.3 and 100.0 mg/L were investigated in the main study. The test solutions used in the test was prepared by mechanical dispersion without using any solubilising agents. An appropriate amount of test item was dissolved in the dilution water (OECD medium) in order to obtain the concentration of 100 mg/L. The further test solutions were prepared by appropriate dilution of this stock solution. After the formulation procedure algal cells were immediately introduced into the test solutions. Untreated control (dilution water (OECD medium) without addition of the test item) ran parallel in the test and a positive control with potassium dichromate was performed. The components of the test item (Phosphoric acid mono(2-ethylhexyl) ester, Pyrophosphoric acid di(2-ethylhexyl) ester and Phosphoric acid bis(2-ethylhexyl) ester) were used for the quantitation of the test item. Samples were taken from each test concentrations and from the control at the start (0 hours) and at the end of exposure (72 hours) and analyzed using a HPLC- MS method. Measured concentrations of these components were in the range of 71 – 103 % of the nominal at the start and 53 – 119 % at the end of the test. The corresponding measured geometric mean test item concentrations were: 0.92, 3.38, 10.77, 33.81 and 103.99 mg/L. Biological results are based on the measured geometric mean test item concentrations as the analytically measured test item concentrations were not within the range of ± 20 % of the nominal over the test period of 72 hours. The 24h-ErC50 and the EyC50 of potassium dichromate was determined to be 0.82 and 0.51 mg/L respectively. The validity criteria were fulfilled. After 72 hours the ErC10 was determined to be 15.50 mg/L and the ErC50 was determined to be 34.63 mg/L.