Phosphoric acid, 2-ethylhexyl ester, ammonium salt

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:

13-June-17 to 16-Feb-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in acordance with international guideines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: confidential
- Expiration date of the lot/batch: 01 Jan 2019
- Purity test date: N/D

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, dark, cool (2-8 °C)
- Stability under test conditions: sufficient for the test purpose
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All tested concentrations were analysed at 0 and 72 hours, the concentrations were 0.954, 3.05, 9.77, 31.3, 100 mg/L.
- Sampling method: Analytical samples were taken at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions from all loading rates and control. For each sampling also a retain sample was taken. 500 µL samples were taken and stabilized with 500 µL methanol.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accomodated fractions were used as per the OECD guidance on testing and assessment no. 23. Each concentration was separately prepared. The prerequisite amount of test item was added to the test vessel containing the test medium and stirred for 48 h in the dark at room temperature. After the settling the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the stock solution. Algae were added to each solution individually resulting in nominal cell densities of 0.5 × 10^4 cells per mL in each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel. The preparation of the test solutions is shown below.
- Eluate: N/A
- Differential loading: N/A
- Controls: AAP medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The solutions were clear and transparent and foaming was observed for 100 mg/L and 31.3 mg/L. Subsequently the undissolved test item was allowed to sediment for 4 hours until the phases had separated. For 100 mg/L and 31.3 mg/L the Tyndall’s effect could be observed even after 4 hours of settling. The extension of the separation phase did not lead to the removal of the Tyndall’s effect (i.e. particulate matter) in 100 mg/L and 31.3 mg/L test vessels up to 48 hours. Further, there was no substantial improvement to the level of Tyndall’s effect shown at 4 hours compared to 24 or 48 hours. and therefore 4 hours was seen as an optimal settling time for the WAF. All other concentrations were free from particulate matter as indicated by the lack of Tyndall's effect.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from the commercial supplier.
Culture conditions were as follows:
Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 µEm-2s-1)
Temperature: 21 - 24 °C
Culture flasks: 100 mL Erlenmeyer flasks
CO2 supply by shaking on a rotating shaker, approximately 105 rpm
Cells from this semi-continuous liquid stock culture were used for the test.

ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test
- 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:

OECD 201 guideline duration.

Post exposure observation period:

none

Hardness:

N/D

Test temperature:

22.7 to 22.9 °C

pH:

7.00 to 7.96

Dissolved oxygen:

N/D

Salinity:

N/A

Conductivity:

N/D

Nominal and measured concentrations:

Nominal loading rates were, 100 mg/L, 31.3 mg/L, 9.77 mg/L, 3.05 mg/L and 0.954 mg/L (these concentrations are based on test substance added minus water content, as water is not the driver for any observed toxicity). Measured concentrations were 85 to 115 % of nominal. Nominal loading rates were used as analysis was only for verifiaction of correct loading rates as a WAF design was used.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: filled with 50 mL medium-algae, or medium-algae-test item.
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 0.5 x 10^4
- Control end cells density: 65.18 x 10^4
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): no vehicle was used and no vehicle controls were required.

GROWTH MEDIUM
- Standard medium used: yes (AAP medium, OECD 201)
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory prepared.
- Total organic carbon: not relevant/required
- Particulate matter: not relevant/required
- Metals: not relevant
- Pesticides: not relevant
- Chlorine: not relevant/required
- Alkalinity: not relevant/required
- Ca/mg ratio: not relevant/required
- Conductivity: not relevant/required
- Culture medium different from test medium: no
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72.

OTHER TEST CONDITIONS
- Sterile test conditions: not required
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 82.6 to 93.6 µEm-2s-1 (-7.0 % to +5.4 % of mean) which was within ± 15 % of variation as specified by OECD 201.
- Salinity (for marine algae): not relevant

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter, calibrated using cell counter Neubauer chamber.
- Chlorophyll measurement: none
- Other: none

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: 5 concentrations used in accordance with OECD guideline 201
- Range finding study: Yes, conducted at 0, 7.5 and 75 mg/L.
- Test concentrations: 0, 0.954, 3.05, 9.77, 31.3 and 100 mg/L
- Results used to determine the conditions for the definitive study: no effect at 7.5 or 75 mg/L were observed in the range finding test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used as the positive control.

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield (migrated information)

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield (migrated information)

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield (migrated information)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: -
- Any stimulation of growth found in any treatment: there was a mild increase in cell number and growth rate between 0.954 to 9.77 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: loading rates were used.
- Effect concentrations exceeding solubility of substance in test medium: some particulate matter was observed at the highest concentration of 100 mg/L. However, this is the limit concentration as advised by the OECD 201 guideline and, minimal to no inhibitory effects were seen.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.64 mg/L
- Other: NOEC 0.320 mg/L, LOEC 0.800 mg/L

Reported statistics and error estimates:

see table 1 in section below

Table 1:     Percentage inhibition of growth rate

Loading rate	% Inhibition of growth rate^1)
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	7.5	3.3	3.2
Control	-11.0	1.4	-1.9
Control	-16.0	-9.7	-5.4
Control	-5.7	-0.6	0.2
Control	16.3	6.5	5.3
Control	8.8	-0.9	-1.4
Mean	0.0	0.0	0.0
0.954	10.5	-3.2	-3.8
0.954	10.7	-7.0	-3.5
0.954	8.4	-6.2	-4.7
Mean	9.9	-5.5	-4.0
3.05	1.1	-4.7	-4.8
3.05	4.1	-7.9	-5.6
3.05	3.4	-3.2	-4.3
Mean	2.9	-5.3	-4.9
9.77	12.8	-1.9	-4.2
9.77	2.7	-1.3	-4.0
9.77	19.9	5.5	2.7
Mean	11.8	0.8	-1.8
31.3	43.2	6.9	-1.1
31.3	35.3	11.8	1.4
31,3	32.8	6.8	2.6
Mean	37.1	8.5	1.0
100	60.0	18.5	7.9
100	60.0	14.6	3.2
100	41.6	15.1	9.5
Mean	53.9	16.1	6.9 *

* Statistically significant different to the control

¹⁾Values are based on the mean control value

Table 2:     Percentage inhibition of yield

Loading rate	% Inhibition of yield^1)
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	9.4	6.4	10.8
Control	-10.1	13.5	0.6
Control	-29.3	-33.9	-27.0
Control	-11.8	-2.7	0.0
Control	24.5	16.9	20.1
Control	17.4	-0.3	-4.5
Mean	0.0	0.0	0.0
0.954	18.7	-9.9	-19
0.954	19.1	-24.3	-17.4
0.954	15.5	-21.1	-24.4
Mean	17.8	-18.4	-20.3
3.05	3.1	-15.3	-25.4
3.05	8.3	-28.0	-29.9
3.05	7.1	-10.0	-22.0
Mean	6.2	-17.8	-25.8
9.77	22.3	-5.3	-21.3
9.77	5.9	-3.3	-20.2
9.77	32.7	17.4	13.2
Mean	20.3	2.9	-9.4
31.3	60.2	21.2	-4.4
31.3	51.8	33.3	7.6
31.3	49.0	21.1	12.9
Mean	53.7	25.2	5.4
100	75.4	46.7	32.7
100	75.4	39.2	15.1
100	58.6	40.2	37.8
Mean	69.8	42.0	28.5 *

* Statistically significant different to the control

¹⁾Values are based on the mean control value

Table 3:     pH measurements during the test

Loading rate	t = 0 h	t = 72 h
[mg/L]
Control	7.00	7.75
0.954	7.00	7.96
3.05	7.04	7.95
9.77	7.02	7.96
31.3	7.00	7.93
100	7.02	7.83

Validity criteria fulfilled:

yes

Conclusions:

A test was conducted to determine the effects of the Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts on the growth of the single cell green alga Pseudokirchneriella subcapitata. The study was conducted in accordance with the OECD guideline 201 the “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23. There were no deviations from the guidance or study plan, and all validity criteria were met.

The NOELR for yield and growth rate was 31.3 mg/L. The EL10-, EL20- and EL50-value for growth rate and the EL50-value for yield were considered to be > 100 mg/L (nominal). Due to an inhibition of yield below 50 % and a missing loading rate response relation no reliable values were calculable and the EL10- and EL20-value for yield was not determined.

Executive summary:

The objective of this study was to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata, to determine the no observed effect loading rate (NOELR), to determine the lowest observed effect loading rate (LOELR) and to determine the effect loading rate (EL10, 20, 50), where possible.

The study was conducted in accordance with the OECD guideline 201 the “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.

The definitive test concentrations were, 0.954, 3.05, 9.77, 31.3 and 100 mg/L (all concentrations are based on test item added minus its water content as water is deemed to not contribute to the toxic effects observed, if any). WAFs were prepared at each concentration by adding a prerequisite amount of test item to the test vessel, stirring for 48 hours in the dark at room temperature and then leaving any particulate to settle for 4 hours. Some particles were still in the upper two concentrations as indicated by the Tyndall's effect but this did not impact the integrity of the study. Furthermore, longer settling periods did not reduce the presence of particulate in these vessels. Water quality parameters were measured at the start and end of the test or daily. Analytical confirmation of the test substance was performed at each concentration at 0 and 72 hours. All water quality parameters and study validity criteria were met as per OECD guideline 201. A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism.

The NOELR for yield and growth rate was 31.3 mg/L. Due to an inhibition of yield below 50 % and a missing loading rate response relation no reliable values were calculable and the EL10- and EL20-value for yield was not determined. The EL10-, EL20- and EL50-value for growth rate and the EL50-value for yield were considered to be > 100 mg/L (nominal).

Description of key information

72h EL50 (growth rate) > 100 mg/L, 72 h EC10 (growth rate) > 100 mg/L (Pseudokirchnerella subcapitata), OECD 201 (2011), ; Schuster A.-K. (2017).

Additional information

One Key Study is alvailable for the endpoint which assessed the toxicity of the test item to a freshwater alga.

The study was conducted in accordance with the OECD guideline 201 the “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.

The objective of this study was to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata, to determine the no observed effect loading rate (NOELR), to determine the lowest observed effect loading rate (LOELR) and to determine the effect loading rate (EL10, 20, 50), where possible.  

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.

The definitive test concentrations were, 0.954, 3.05, 9.77, 31.3 and 100 mg/L (all concentrations are based on test item added minus its water content as water is deemed to not contribute to the toxic effects observed, if any). WAFs were prepared at each concentration by adding a prerequisite amount of test item to the test vessel, stirring for 48 hours in the dark at room temperature and then leaving any particulate to settle for 4 hours. Some particles were still in the upper two concentrations as indicated by the Tyndall's effect but this did not impact the integrity of the study. Furthermore, longer settling periods did not reduce the presence of particulate in these vessels. Water quality parameters were measured at the start and end of the test or daily. Analytical confirmation of the test substance was performed at each concentration at 0 and 72 hours. All water quality parameters and study validity criteria were met as per OECD guideline 201. A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism.

The EL10-, EL20- and EL50-value for growth rate were considered to be > 100 mg/L (nominal). As the effect level is greater than the regulatory limit concentration of 100 mg/L, no hazard to aquatic algae and cyanobacteria has been identified.