Trimethyl[2-(phosphonooxy)ethyl]ammonium chloride, calcium salt (1:1)

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:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.3, 1, 3.1, 9.8, 31.3 and 100 mg/L
- Sampling method: at the beginning (0 h) and at the end (72 h) of the test.

Vehicle:

yes

Remarks:

OECD medium

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662, UTEX 1648
- Method of cultivation: An OECD culture medium was diluted to produce the biomass of 1 E04 cells/ml and then sub-cultured continuously at intervals of 3 to 4 days. For pre-cultivation, the test algae were inoculated into the OECD culture medium at a biomass of 0.5-1.0 E04 cells/ml 2 to 4 days prior to study initiation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

The OECD medium was in accordance with the OECD guideline 201.

Test temperature:

23.1 - 24.2 ºC

pH:

7.50 - 9.08

Salinity:

The OECD medium was in accordance with the OECD guideline 201.

Nominal and measured concentrations:

Nominal: 0.3, 1, 3.1, 9.8, 31.3 and 100 mg/L
Measured: 0.3, 0.4, 0.3, 0.8, 1.7 and 4.4 mg/mL

Details on test conditions:

TEST SYSTEM
- Initial cells density: 0.5-1 E04 cells/mL

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

OTHER TEST CONDITIONS
- Light intensity and quality: 4440 - 8880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Morphological changes of algal cells were observed including expansion, agregation, atrophy and decoloration at 72 h after exposure.

TEST CONCENTRATIONS
- Range finding study:
- Test concentrations: 0.1, 1 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The percent inhibition of growth rates was 13.5% for the average specific growth rate and 51.4% for the yield at the highest dose of 100 mg/L.

Reference substance (positive control):

not specified

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:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Calcium hydrogenorthophosphate was insoluble in the OECD culture medium and therefore the concentration of the test substance was detected to be significantly low during the test. The measured concentrations of the test substance were 0.3, 0.4, 0.3, 0.8, 1.7 and 4.4 mg/L (nominal concnetrations: 0.3, 1.0, 3.1, 9.8, 31.3, and 100 mg/L). At 72 h, the test substance in the test solution was not detected at 0.3, 1.0 and 3.1 mg/L (nominal), but its concentrations at 9.8, 31.3 and 100 mg/l (nominal) were estimated to be 0.3, 1.6 and 4.7 mg/L.

Reported statistics and error estimates:

Statistical analysis was not performed, since less than 50% of growth inhibition at 100 mg/L (nominal) was observed in the test and therefore, the EC50 values were expressed as above the test concentration.

Algal cells were observed to maintain a similar shape to the control in all test solutions, and microorganisms other than algae were not observed.

The growth curves for cell density measured in triplicate every 24 hour in each concentration of the tests substance are shown in the graph attached.

The 72 h EC50 was estimated to be > 100 mg/L (measured concentration: > 4.4 mg/L) using the average specific growth rate and yield.

Validity criteria fulfilled:

not specified

Conclusions:

The 72h-EC50 of calcium hydrogenorthophosphate in Pseudokirchneriella subcapitata was found to be higher than 100 mg/L (nominal) and the NOEC was determined to be 100 mg/L, both based on growth rate.

Executive summary:

A freshwater algae growth inhibition test was performed on calcium hydrogenorthophosphate according to OECD Guideline 201, following GLP. A range finding study performed at concentrations of 0.1, 1, 10 and 100 mg/L test item and no toxicity was observed at the maximum dose. Therefore, the definitive test was performed exposing Pseudokirchneriella subcapitata to the nominal concentrations of 0 (control), 0.3, 1, 3.1, 9.8, 31.3 and 100 mg/L for 72 hours. The analysis of the test substance in the test solution was performed at the beginning (0 h) and at the end of the test (72 h) using inductively coupled plasma (ICP). The 72h-EC50 of test item in Pseudokirchneriella subcapitata was found to be higher than 100 mg/L(nominal) and the NOEC was determined to be 100 mg/L, both based on growth rate.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Wako Pure Chemical Industries, Ltd., Lot No.: PAR1681.
- Purity: 100.2%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1000, 320, 100, 32, 10, 3.2 and 1.0 mg/L
- Sampling method: Test concentrations were mesured at the start and the end of test.

Vehicle:

yes

Remarks:

OECD medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test substance was diluted with OECD medium. Test substance was stored in freezer. The stability of the chemical was confirmed by IR absorption spectrum. Under the stock condition, IR spectrum of the test substance at the end of test was same at the start.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapita
- Strain: ATCC22662
- Source (laboratory, culture collection): Obtained from subcultured in Kureha Special Laboratory Co., Ltd.
- Method of cultivation: sterile

ACCLIMATION
- Acclimation period: 4 days

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

The OECD medium was in accordance with the OECD guideline 201.

Test temperature:

23 ± 2 ºC

Salinity:

The OECD medium was in accordance with the OECD guideline 201.

Nominal and measured concentrations:

1000, 320, 100, 32, 10, 3.2 and 1.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL of medium in a 300 mL glass
- Aeration: agitation 100 rpm
- Initial cells density: 1 E04 cells/ml

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continous ilumination
- Light intensity and quality: 4000-5000 lux

TEST CONCENTRATIONS
- Test concentrations: 1, 3.2, 10, 32, 100, 320 and 1000 mg/l.

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

NOECr

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Test concentratoins were measured at the start and at the end of test using by HPLC. All of them, the deviation from the nominals were less than +/- 10%.

Nominal Conc. [mg/l]	Measured Conc. [mg/L]		% of Nominal Conc.
	0 h	72 h	0 h	72 h
Control	N.D.	N.D.	--	--
1.0	0.8	1.0	80	100
3.2	3.0	3.1	94	97
10	10.0	10.1	100	101
32	31.3	32.1	98	100
100	103	108	103	108
320	328	345	103	108
1000	1070	1070	107	107

- Growth curves: exponential growth phase was kept during 72 hours.

- Calculation of toxic value: Nominal concentration.

Validity criteria fulfilled:

not specified

Conclusions:

The 72h-EC50 (based on growth rate reduction) of choline chloride in Pseudokichneriella subcapitata was greater than 1000 mg/L and the 72h-NOEC was 1000 mg/L.

Executive summary:

A freshwater algae growth inhibition test was performed with choline chloride according to OECD Guideline 201, following GLP. The following test item concentrations 0 (control), 1, 3.2, 32, 100, 320 and 1000 mg/L were tested. Pseudokirchneriella subcapitata (1 E04 cells/ml) were exposed to the different concentrations of the test item or the control for 72 h. The analysis of the test substance in the test solution (OECD medium) was performed at the beginning and the end of the 72 h of exposure by HPLC. The 72h-EC50 (based on growth rate reduction) of choline chloride in Pseudokichneriella subcapitata was greater than 1000 mg/L and the 72h-NOEC was 1000 mg/L.

Description of key information

The available data refer to the degradation products of calcium phosphorylcholine chloride (choline and inorganic phosphate):

- Key study. OECD 201. GLP study. The 72h-EC50 (based on growth rate reduction) of choline chloride in Pseudokichneriella subcapitata was greater than 1000 mg/L and the 72h-NOEC was 1000 mg/L.

- Key study. OECD 201. GLP study. The 72h-EC50 of calcium hydrogenorthophosphate in Pseudokirchneriella subcapitata was found to be higher than 100 mg/L (nominal) and the NOEC was determined to be 100 mg/L, both based on growth rate.

Based on this information, it can be concluded that the substance phosphoryl choline chloride has a 72h-EC50 in Pseudokirchneriella subcapitata higher than 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

- Key study. A freshwater algae growth inhibition test was performed with choline chloride according to OECD Guideline 201, following GLP. The following test item concentrations 0 (control), 1, 3.2, 32, 100, 320 and 1000 mg/L were tested. Pseudokirchneriella subcapitata (1 E04 cells/ml) were exposed to the different concentrations of the test item or the control for 72 h. The analysis of the test substance in the test solution (OECD medium) was performed at the beginning and the end of the 72 h of exposure by HPLC. The 72h-EC50 (based on growth rate reduction) of choline chloride in Pseudokichneriella subcapitata was greater than 1000 mg/L and the 72h-NOEC was 1000 mg/L.

- Key study. A freshwater algae growth inhibition test was performed on calcium hydrogenorthophosphate according to OECD Guideline 201, following GLP. A range finding study performed at concentrations of 0.1, 1, 10 and 100 mg/L test item and no toxicity was observed at the maximum dose. Therefore, the definitive test was performed exposing Pseudokirchneriella subcapitata to the nominal concentrations of 0 (control), 0.3, 1, 3.1, 9.8, 31.3 and 100 mg/L for 72 hours. The analysis of the test substance in the test solution was performed at the beginning (0 h) and at the end of the test (72 h) using inductively coupled plasma (ICP). The 72h-EC50 of test item in Pseudokirchneriella subcapitata was found to be higher than 100 mg/L(nominal) and the NOEC was determined to be 100 mg/L, both based on growth rate.