Octylphosphonic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2011-27-07 to 2013-03-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with international standard guidelines under GLP conditions. The pH values of the post-study are missing in the final report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-08-31

Test material

Test material information

Constituent 1

Reference substance name:

Octylphosphonic acid

EC Number:

225-218-5

EC Name:

Octylphosphonic acid

Cas Number:

4724-48-5

Molecular formula:

C8H19O3P

IUPAC Name:

octylphosphonic acid

Test material form:

other: granular solid

Details on test material:

- Name of test material (as cited in study report):Octylphosphinic acid (OPA)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control (replicate R1-R6 pooled) and each test group (replicates R1-R3 pooled) at 0 and 72 hours for quantitative analysis. All o-hour samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary

Test solutions

Vehicle:

no

Details on test solutions:

Amount of test item (100 and 32 mg) were each separely dissolved in the culture medium with the aid of ultrasonication for approximately 30 minutes and the volumes adjusted to 1 litter to give a stock solutions of 100 and 32 mg/L. A series of dilutions was made from these stock solutions of 100 mg and 32 mg/L. A serie of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (11.7 mL) to give the required test concentration of 1.0; 3.2, 10, 32 and 100 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The concentrations and stability of the test item in the test preparation were verified by chemical analysis at 0 and 72 hours.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name:Pseudokirchneriella subcapitata
- Strain:CCAP 278/4
- Source (laboratory, culture collection):Liquids cultures were obtained from the culture collection of Algae and protozoa (CCAP°, SAMS research services Ltd, Scottish Marine Institute, Oban, Argyll Scotland
- Age of inoculum (at test initiation): No data
- Method of cultivation: Masters cultures were maintained in the laboratory by the periodic replenishment of culture medium, and maintained under constant aeration and illumination at 21°C ±1°C

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10e3 cells/mL. The flask were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24°C ± 1°C until algal cell density was approximately 10e4-10e5 cells/mL.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.83 x 10e5 cells per mL. Inoculation of 900 mL of test medium with 11.7 mL of this algal suspension gave an initial nominal cell density of 5 x 10e3 cells per mL and had no significant dilution effect on the final test concentration.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

No data

Test temperature:

24°C ± 1°C

pH:

The pH values of the control culture was observed to increase from pH = 8.2 at o-hour to pH = 8.3 at 72-hour . The pH deviation in the control cultures was less than 1.5 units after 72 hours and therefore was within the limits given in the guidelines

A concentration dependent decline in pH was observed with increasing test concentration at 0 hours in the range of pH = 8.1 at 1.0 mg/L through to pH = 3.7 at 100 mg/L. This was considered to be due to an intrinsic property of the test item and hence no adjustment was made to the pH prior to exposure

Dissolved oxygen:

No data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentration: 0, 1.0, 3.2, 10, 32, 100 mg/L
Measured concentration at 0h: 0.745, 0.677, 2.85, 9.15, 30.7, 92.2 mg/L (*)
Measured concentration at 72h: 0, 0.916, 3.05, 9.73, 32.7, 103 mg/L

(*): at 0h, analysis of the sample showed a measured test concentration of 75% of nominal concentration. Analysis of duplicate sample showed a measured concentration of 68% of nominal concentration. Inspection of the data could find no cause for low measured test concentration. Given that a concentration of 92% of nominal concentration was observed at 72 hours, it was considered that the result obtained from o-hour analysis were erroneous. This was considered to have had no adverse effect on the outcome of the test given that the 1.0 mg/L test concentration was below the NOEC.
As such,it was considered appropriate to calculate the results based on nominal test concentration only

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flask
- Type (delete if not applicable): closed (Polyurethane foam bungs)
- Material, size, headspace, fill volume: fill at 100 mL
- Aeration: constantly shaken at approximately 150 rpm during 72h
- Initial cells density: 5x 10e3
- Control end cells density: At 0h, 24h, 47h, 72h
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Incubation in INFORS Multitron version 2 incubator

GROWTH MEDIUM
Medium used as describe in guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water (Elga Optima 15 + or Elga Purelab Option R-15 BP)

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: yes. The pH was adjusted at 7.5 ± 0.1 for the culture medium prior to introduce test item
- Photoperiod: constant illumination
- Light intensity and quality: approximately 7000 lux provided by warm white lighting (380-730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:10
- Justification for using less concentrations than requested by guideline:
- Range finding study: YES
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No effects were observed on growth at 0.1, 1 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 34-44 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 20-26 mg/L

Details on results:

Cell density values determined at each sapling time and pH values at 0 and 72 hours are given in table 2.
Daily specific growth rate for the control cultures are given in table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in table 4.

Validation criteria:
The following data show that the cell concentration of the control cultures increased by a factor of 176 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
Mean cell density of control at 0 hours : 2.50 x 10e3 cells per mL
Mean cell density of control at 72 hours : 9.28 x 10e5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% 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 (0-72h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) gave the following results:
ErC50 (0-72h): 1.4 mg/L (1.2-1.7 mg/L)
EyC50 (0-72h): 0.59 mg/L (0.53-0.65 mg/L)

NOEC growth rate: 0.25 mg/L
NOEC yield: 0.25 mg/L
LOEC growth rate: 0.50 mg/L
LOEC yield: 0.50 mg/L

The results 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 severals 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 concentration to determine any statistical significant differences between the test and the control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999-2001)

Any other information on results incl. tables

Table 2: Cell Densities and pH values in the definitive test

 

Nominal concentration (mg/L)		pH	Cell densities * (cells per mL)				pH
		0h	0h	24h	47h	72h	72h
Control	R1	8.2	5.04.E+3	3.02.E+4	1.49.E+5	7.24.E+5	8.3
	R2		5.19.E+3	3.00.E+4	1.74.E+5	9.36.E+5
	R3		5.02.E+3	2.73.E+4	1.71.E+5	8.55.E+5
	R4		5.82.E+3	3.46.E+4	1.75.E+5	1.06.E+6
	R5		5.09.E+3	3.12.E+4	1.34.E+5	8.83.E+5
	R6		5.04.E+3	3.51.E+4	1.21.E+5	1.11 E+6
	Mean		5.20.E+3	3.14.E+4	1.54.E+5	9.28.E+5
1.0	R1	8.1	5.23.E+3	3.16.E+4	1.64.E+5	1.00 E+6	8.2
	R2		5.06.E+3	3.38.E+4	1.95.E+5	1.03 E+6
	R3		5.38.E+3	3.18.E+4	1.50.E+5	1.01 E+6
	Mean		5.23.E+3	3.24.E+4	1.69.E+5	1.01 E+6
3.2	R1	7.9	5.22.E+3	3.74.E+4	1.67.E+5	8.66.E+5	8.1
	R2		5.02.E+3	3.57.E+4	1.78.E+5	1.17 E+6
	R3		5.09.E+3	3.02.E+4	1.43.E+5	9.19.E+5
	Mean		5.11.E+3	3.44.E+4	1.63.E+5	9.85.E+5
10	R1	7.7	5.15.E+3	3.62.E+4	1.54.E+5	9.24.E+5	7.9
	R2		5.22.E+3	3.69.E+4	1.74.E+5	8.30.E+5
	R3		5.06.E+3	3.83.E+4	1.90.E+5	8.92.E+5
	Mean		5.14.E+3	3.72.E+4	1.72.E+5	8.82.E+5
32	R1	7.3	5.30.E+3	2.26.E+4	7.85.E+4	3.32.E+5	7.3
	R2		5.14.E+3	1.98.E+4	5.07.E+4	1.79.E+5
	R3		5.30.E+3	1.93.E+4	4.45.E+4	1.21.E+5
	Mean		5.25.E+3	2.05.E+4	5.79.E+4	2.11.E+5
100	R1	3.7	5.06.E+3	1.40.E+4	5.38.E+3	6.98.E+3	3.5
	R2		5.03.E+3	1.55.E+4	1.12.E+4	4.01.E+3
	R3		5.07.E+3	1.66.E+4	7.54.E+3	6.41.E+3
	Mean		5.06.E+3	1.54.E+4	8.04.E+3	5.80.E+3

* cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1-R6 = Replicate 1 to 6

 

 

Table 3: Daily specific growth rates for the control cultures in the definitive test

 

		Daily specific growth rate (cells/mL/hour)
		Day 0-1	Day 1-2	Day 2-3
Control	R1	0.075	0.069	0.063
	R2	0.075	0.076	0.067
	R3	0.071	0.080	0.064
	R4	0.081	0.071	0.072
	R5	0.076	0.063	0.075
	R6	0.081	0.054	0.089
	Mean	0.077	0.069	0.072

R1-R6 = Replicate 1 to 6

Table 4: Inhibition of growth rate and yield in the definitive test

 

Nominal concentration (mg/L)		Growth rate (cell/mL/hour)		Yield (Cell/mL)
		0-72 h	% Inhibition +	0-72 h	% Inhibition +*
Control	R1	0.069		7.19.E+05
	R2	0.073		9.31.E+05
	R3	0.071		8.50.E+05
	R4	0.074	-	1.06.E+06	-
	R5	0.074		8.78.E+05
	R6	0.072		1.11.E+06
	Mean	0.075		9.23.E+05
	SD	0.002		1.42.E+05
1.0	R1	0.074	[3]	9.99.E+05
	R2	0.074	[3]	1.03.E+06
	R3	0.074	[3]	1.00.E+06
	Mean	0.074	[3]	1.01.E+06	[9]
	SD	0.000		1.49.E+04
3.2	R1	0.072	0	8.61.E+05
	R2	0.076	[6]	1.16.E+06
	R3	0.072	0	9.14.E+05
	Mean	0.073	[2]	9.80 .E+05	[6]
	SD	0.002		1.62 .E+05
10	R1	0.072	0	9.19 .E+05
	R2	0.071	1	8.25 .E+05
	R3	0.072	0	8.87 .E+05
	Mean	0.072	0	8.77 .E+05	5
	SD	0.001		4.77 .E+04
32	R1	0.058	19	3.27 .E+05
	R2	0.050	31	1.73 .E+05
	R3	0.044	39	1.16 .E+05
	Mean	0.051	30	2.05 .E+05	78
	SD	0.007		1.09 .E+05
100	R1	0.005	93	1.92 .E+03
	R2	-0.003	104	-1.02 .E+03
	R3	0.003	96	1.34 .E+03
	Mean	0.002	98	7.44 .E+02	100
	SD	0.004		1.56 .E+03

+ In accordance with the OECD test guideline inhibition of growth was calculated based on the 0 -Hour measured cell densities whilst inhibition of yield was calculated based on a nominal cell density of 5.00 E+03 cells/mL

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

R1 - R6 = Replicates 1 to 6

[Increase in growth as compared to controls]

Table 5: Cell Densities and percentage inhibition of Growth from the post-study experiment

Nominal Concentration (mg/L)		Cell Densities * (Cells per mL)		Inhibition Values (%)**
		0 hours	72 hours	Growth Rate	Yield
Control	R1	5.68 E+03	6.68 E+05	-	-
	R2	5.01 E+03	4.67 E+05
	Mean	5.34 E+03	5.67 E+05
1.0	R1	5.23 E+03	7.95 E+05	[6]	[31]
	R2	5.17 E+03	6.87 E+05
	Mean	5.20 E+03	7.41 E+05
10	R1	5.38 E+03	5.70 E+05	[2]	[12]
	R2	5.26 E+03	6.95 E+05
	Mean	5.32 E+03	6.32 E+05
100	R1	7.14 E+03	5.89 E+04	57	92
	R2	6.66 E+03	4.30 E+04
	Mean	6.90 E+03	5.10 E+04

*Cell densities represent the mean number of cell per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

**Inhibition values for growth rate and yield determined based on the 0 -hour measured cell densities

R1 and R2 = Replicate 1 and Replicate 2

[Increase in growth compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-hour period and gave the following result
s:
ErC10 : 23 mg/L
ErC50 : 40 mg/L with 95% confidence limits of 35-44 mg/L
EyC50 : 23 mg/L with 95% confidence limits of 20-26 mg/L

NOEC (growth rate) = 10 mg/L
NOEC (Yield) = 10 mg/L
LOEC Growth rate: 32 mg/L
LOEC Yield = 32 mg/L

Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to OPA at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L under static conditions in accordance with the OECD 201, after a range finding study at nominal concentration of 0.1, 1.0, 10 and 100 mg/L

Analytical verification showed an acceptable correlation between the nominal and the measured concentration.

The results were based on nominal concentration

The following results were determined:

NOEC Growth Rate and yield = 10 mg/L

EC10 Growth rate= 23 mg/L

EC 50 Growth rate= 40 mg/L with confidence limits of 35 -44 mg/L

EC 50 Yield = 23 mg/L with confidence limits of 20 -26 mg/L

All validity criteria were met, the test can be considered to be valid