Ethyltriphenylphosphonium acetate

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:

19 February 2018 - 24 March 2018

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
An amount of test item (50.5 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 500 mL to give a nominal 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 3.2, 1.0, 0.32 and 0.10 mg/L. An aliquot (500 mL) of each of the 0.10, 0.32, 1.0, 3.2 and 10 mg/L stock solutions was separately inoculated with algal suspension (2.5 mL) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium

ACCLIMATION
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ±1 ºC

pH:

7.4 -7.8 at test start, 7.8 - 8.4 at test end

Nominal and measured concentrations:

0, 0.10, 0.32, 1.0, 3.2 and 10 mg/L (nominal)
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89% to 98% of nominal and so the results are based on the nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks containing 100 mL of test preparation
- Aeration: constantly shaken at approximately 150 rpm for 72 hours.
- Type (delete if not applicable): closed
- Initial cells density: 5.00E03 cells/mL
- Control end cells density: 1.73E+06 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture medium different from test medium: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no
- Other:
Samples were taken at 19, 42 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00E03 cells/mL) was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.



TEST CONCENTRATIONS
- Spacing factor for test concentrations: sqrt(10)
- Range finding study
- Test concentrations: range-finding test: 0, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
The results showed no effect on growth at the test concentration of 0.10 mg/L. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L.
Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits

Remarks:

3.6 to 5.3 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.13 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.77 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits

Remarks:

0.53 to 1.1 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.10, 0.32, 1.0 and 3.2 mg/L, however cell debris was observed to be present in the test cultures at 10 mg/L.
- At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.10 and 0.32 mg/L test cultures were observed to be green dispersions. The 1.0 mg/L test cultures were light green dispersions; the 3.2 mg/L test cultures were extremely pale green dispersions whilst the 10 mg/L test cultures were clear colorless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validity Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 347 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours: 5.00E03 cells per mL

Mean cell density of control at 72 hours: 1.73E06 cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.082	-	1.85E+06	-
	R2	0.081		1.71E+06
	R3	0.082		1.79E+06
	R4	0.081		1.75E+06
	R5	0.081		1.74E+06
	R6	0.080		1.54E+06
	Mean	0.081		1.73E+06
	SD	0.001		1.04E+05
0.10	R1	0.080	1	1.62E+06
	R2	0.080	1	1.64E+06
	R3	0.079	2	1.50E+06
	Mean	0.080	1	1.59E+06	8
	SD	0.001		7.56E+04
0.32	R1	0.077	5	1.26E+06
	R2	0.076	6	1.22E+06
	R3	0.077	5	1.30E+06
	Mean	0.077	5	1.26E+06	27
	SD	0.001		3.94E+04
1.0	R1	0.071	12	8.14E+05
	R2	0.070	14	7.56E+05
	R3	0.069	15	6.93E+05
	Mean	0.070	14	7.54E+05	56
	SD	0.001		6.03E+04
3.2	R1	0.056	31	2.75E+05
	R2	0.056	31	2.71E+05
	R3	0.053	35	2.24E+05
	Mean	0.055	32	2.57E+05	85
	SD	0.002		2.84E+04
10	R1	0.023	72	2.19E+04
	R2	0.010	88	5.24E+03
	R3	0.008	90	3.62E+03
	Mean	0.014	83	1.03E+04	99
	SD	0.008		1.01E+04

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R  = Replicate

SD      =Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

NOEC (growth rate): 0.32 mg/L
ErC10 (0 to 72 hour): 1.2 mg/L
ErC50 (0 to 72 hour): 4.4 mg/L; 95% confidence limits 3.6 to 5.3 mg/L
 
NOEC (yield): 0.1 mg/L
EyC10 (0 to 72 hour): 0.13 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.53 to 1.1 mg/L

Executive summary:

A study was performed to assess the effect of the test item ETPPAAc on the growth of the green alga Pseudokirchneriellasubcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89% to 98% of nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:

 

NOEC (growth rate): 0.32 mg/L

ErC10 (0 to 72 hour): 1.2 mg/L

ErC50 (0 to 72 hour): 4.4 mg/L; 95% confidence limits 3.6 to 5.3 mg/L

 

NOEC (yield): 0.1 mg/L

EyC10 (0 to 72 hour): 0.13 mg/L

EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.53 to 1.1 mg/L

 

Description of key information

NOEC (growth rate)      :         0.32 mg/L

ErC10 (0 to 72 hour)     :          1.2 mg/L

ErC20 (0 to 72 hour)     :          1.9 mg/L

ErC50 (0 to 72 hour)     :          4.4 mg/L; 95% confidence limits 3.6 to 5.3 mg/L

OECD TG 201, Pseudokirchneriella subcapitata, static

 

Key value for chemical safety assessment

EC50 for freshwater algae:

4.4 mg/L

EC10 or NOEC for freshwater algae:

1.2 mg/L

Additional information

A study was performed to assess the effect of ETPPAAc on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of0.10,0.32,1.0,3.2and10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^® Multisizer Particle Counter.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89% to 98% of nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:

NOEC (growth rate)      :         0.32 mg/L

ErC10 (0 to 72 hour)     :          1.2 mg/L

ErC20 (0 to 72 hour)     :          1.9 mg/L

ErC50 (0 to 72 hour)     :          4.4 mg/L; 95% confidence limits 3.6 to 5.3 mg/L

NOEC (yield)                 :         0.1 mg/L

EyC10 (0 to 72 hour)     :         0.13 mg/L

EyC20 (0 to 72 hour)     :          0.25 mg/L

EyC50 (0 to 72 hour)     :          0.77 mg/L; 95% confidence limits 0.53 to 1.1 mg/L