Phosphoric acid, butyl ester, sodium salt

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

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:

EC10

Effect conc.:

76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL = 65 - 83 %

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-02-13 to 2012-04-16

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)

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:

- Empirical formula: C8H19O4P
- Molecular mass: 210.2 g/mol

Analytical monitoring:

yes

Remarks:

HPLC-UV

Vehicle:

no

Details on test solutions:

- Pre-treatment of test item and preparation of test item concentrations:
A stock solution was prepared using 100.3 mg of the test item which were added to 1 litre of dilution water and treated for 1 h on a magnetic stirrer. The pH was measured to be 6.3.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.556 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton/cellulose stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scene-desmus subspicatus)
- Strain: No. 86.81 SAG
- Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen, Germany.
- Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of ± 2 °C) at a light intensity in the range of 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977)) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre-cultures: Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 24 °C

pH:

at the start of the test: 6.8 - 7.8
at the end of the test: 5.8 - 8.8

Nominal and measured concentrations:

Nominal concentrations: 3.1, 6.3, 12.5, 25, 50 and 100 mg/L
measured: For the nominal concentration of 3.1 mg/L the nominal concentration was 2.5 mg/L after 0 and 2.467 mg/L after 72 h. For the nominal concentration of 100 mg/L the nominal concentration was 103.039 mg/L after 0 and 91.452 mg/L after 72 h.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with cotton/cellulose stoppers
- Material, size, headspace, fill volume: glass, 300 mL, fill volume of 100 mL
- Initial cells density: 5000 cells/mL
- No. of organisms per vessel: 0.556 mL algal inoculum
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Light intensity and quality: 60 to 120 μE x m-2 x s-1 or an equivalent range of 4000 to 8000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

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:

EC10

Effect conc.:

76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL = 65 - 83 %

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Analytical measurements

At the beginning and at the end of the test the test items concentration was determined analytically in the control and all test concentrations. The lowest test item concentration, 3.1 mg/L, slightly dropped below the 80 % recovery limit during the 72-hour exposure period. This is considered as not significant, because the highest determined test item concentration remained stable over the exposure time and, therefore, all results are expressed in terms of nominal concentrations. Effective concentrations ranged from 80.6 % to 103.0 % of nominal values at 0 hours, and from 79.6 % to 91.5 % of nominal values at 72 hours.

Table 1: Analysis

Test item concentration [mg/L]	HPLC values [mg/L]
	0 h	72 h
Control	n.d.	< 0.0421
3.1	2.500	2.467
100	103.039	91.452

 

Table 2: pH values at the start and the end of the study

Test item concentration [mg/L]	pH values
	0 h	72 h
Control	7.8	8.8
3.1	7.8	8.5
6.3	7.8	8.4
12.5	7.7	8.3
25	7.6	8.2
50	7.3	8.0
100	6.8	5.8

 

Table 3: Growth rate dependent on concentration and time

Section-by-section growth rate in Desmodesmus subspicatus as dependent on concentration of the test item and time; Mean: arithmetic mean; Std.Dev.: standard deviation; CV: coefficient of variation (calculated from InputRawData)

Treatment [mg/L]	Control	3.1	6.3	12.5	25	50	100
Mean at 0 h	0	0	0	0	0	0	0
Std. Dev.	0	0	0	0	0	0	0
CV
Mean 0-24 h	1.386	1.386	1.386	1.386	1.386	1.386	1.386
Std. Dev.	0	0	0	0	0	0	0
CV	0	0	0	0	0	0	0
Mean 24-48 h	1.297	1.386	1.426	1.386	1.504	1.381	1.099
Std. Dev.	0.0771	0	0.0680	0	0	0.1257	0
CV	5.943	0	4.770	0	0	9.105	0
Mean 48-72 h	1.859	1.776	1.716	1.616	1.646	1.614	1.259
Std. Dev.	0.1337	0.0653	0.1127	0.0621	0.0124	0.1394	0.1644
CV	7.192	3.676	6.563	3.842	0.754	8.632	13.049

 

 

Table 4: Yield dependent on concentration and time

Yield of Desmodesmus subspicatus cells as dependent on concentration of the test item and time; Mean: arithmetic mean; Std.Dev.: standard deviation; CV: coefficient of variation (calculated from InputRawData)

Treatment [mg/L]	Control	3.1	6.3	12.5	25	50	100
Mean at 0 h	0	0	0	0	0	0	0
Std. Dev.	0	0	0	0	0	0	0
CV
Mean 24 h	1.5	1.5	1.5	1.5	1.5	1.5	1.5
Std. Dev.	0	0	0	0	0	0	0
CV	0	0	0	0	0	0	0
Mean 48 h	6.833	7.5	7.5	7.5	8.5	6.5	5.5
Std. Dev.	0.5774	0	0.5774	0	0	1	0
CV	8.449	0	7.370	0	0	13.333	0
Mean 72 h	46.5	46.833	45.833	39.833	46.167	39.5	20.833
Std. Dev.	2.6458	3.0551	2.0817	2.5166	0.5774	1	3.5119
CV	5.690	6.523	4.542	6.318	1.251	2.532	16.857

 

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 and ErC10 of the test item on green algae were determined to be >100 mg/L and 76 mg/L, respectively. A NOEC of 50 mg/L was calculated.

Executive summary:

A study was performed to assess the adverse effects of the test item on the growth rate of the freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) and OECD Guideline for Testing of Chemicals No. 201 (2006). Exponentially growing algal cells were exposed for a period of 72 hours to a range of nominal concentrations - 3.1, 6.3, 12.5, 25, 50 and 100 mg/L of the test item dissolved in dilution water. To prepare the test media a magnetic stirrer was used. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate, relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration (NOEC). During 72 hours exposure an ErC50 of >100 mg/L and ErC10 of 76 mg/L were measured and a NOEC of 50 mg/L was calculated.

Description of key information

The ErC50 and ErC10 of the source item on green algae were determined to be >100 mg/L and 76 mg/L, respectively. A NOEC of 50 mg/L was calculated.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

76 mg/L

Additional information

No study on aquatic algae with the target substance is available. A read-across to Dibutyl hydrogen phosphate (CAS 107-66-4) was performed to address the endpoint of toxicity to aquatic algae and cyanobacteria.

A study was performed to assess the adverse effects of the test item on the growth rate of the freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) and OECD Guideline for Testing of Chemicals No. 201 (2006). Exponentially growing algal cells were exposed for a period of 72 hours to a range of nominal concentrations - 3.1, 6.3, 12.5, 25, 50 and 100 mg/L of the test item dissolved in dilution water. To prepare the test media a magnetic stirrer was used. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate, relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration (NOEC). During 72 hours exposure an ErC50 of >100 mg/L and ErC10 of 76 mg/L were measured and a NOEC of 50 mg/L was calculated.

In conclusion, the source substance showed no significant toxic effects on the tested animals. Therefore, it is expected that the target substance has no significant toxic effects on fish and the EC50 after 72 h exposure is >100 mg/L and the EC10 is 76 mg/L, respectively.