Diphenyl methylphosphonate

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:

2013-04-15 - 2013-05-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

yes

Details on sampling:

The pH was measured at initiation of the experiment and after the exposure time. The temperature within the incubator was recorded daily. Samples of the control as well as of each test group were taken at 0 and 72 hours for quantitative analysis.

Vehicle:

no

Details on test solutions:

CULTURE MEDIUM
25.5 mg/L NaNO3, 12.16 mg/L MgCl2 x 6H2O, 4.41 mg/L CaCl2 x 2H2O, 14.6 mg/L MgSO4 x 7H2O, 1.044 mg/L K2HPO4, 15.0 mg/L NaHCO3, 0.186 mg/L H3BO3, 0.415 mg/L MnCl2 x 4H2O, 0.00327 mg/L ZnCl2, 0.160 mg/L FeCl3 x 6H2O, 0.00143 mg/L CoCl2 x 6H2O, 0.00726 mg/L Na2MoO4 x 2H2O, 0.000012 mg/L CuCl2 x 2H2O, 0.30 mg/L Na2EDTA x 2H2O
For preparation reverse osmosis purified deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP) was used and the pH was adjusted to 7.5 +/- 0.1 with 0.1 N NaOH or HCl.

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: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottisch Marine Institute, Oban, Argyll, Scotland

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period described.

Hardness:

No details available.

Test temperature:

24 +/- 1 °C

pH:

Control: 7.5 (start) - 8.0 (end)
1.0 mg/L: 7.4 (start) - 7.9 (end)
3.2 mg/L: 7.4 (start) - 7.9 (end)
10 mg/L: 7.3 (start) - 7.8 (end)
32 mg/L: 7.3 (start) - 7.7 (end)
100 mg/L: 7.2 (start) - 7.6 (end)

Dissolved oxygen:

No details available.

Salinity:

Not applicable.

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed
- Fill volume: 100 mL
- Initial cells density: 5 x 10E3 cells/mL
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 6 replicates

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination (approx. 7000 lux)
- Light intensity and quality: warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Coulter Multisizer Particle Counter

RANGE FINDING STUDY
- Test concentrations: 0, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No effect was observed at test concentration of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 12 - 19 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

No abnormalities in the control and test cultures were detected after 72 hours at the test concentrations of 1.0 and 3.2 mg/L. Few intact cells and cell debris was observed to be present in the test cultures at 10 and 32 mg/L whilst almost no intact cells were observed in the 100 mg/L test cultures.

Results with reference substance (positive control):

- ErC50(72h): 1.2 mg/L; 95 % CL: 1.0 - 1.3 mg/L / NOECr: 0.25 mg/L / LOECr: 0.50 mg/L
- EyC50(72h): 0.52 mg/L; 95 % CL: 0.45 - 0.62 mg/L / NOECy: 0.25 mg/L / LOECr: 0.50 mg/L
These results were within the normal ranges for the reference substance potassium dichromate.

Reported statistics and error estimates:

- One way analysis of variance incorporating Bartlett´s test for homogeneity of variance (Sokal and Rohlf, 1981)
- Dunnett´s multiple comparison procedure (Dunnett, 1955)

Table 1. Cell densities and percentage inhibition of growth - Range finding test

		Cell Densities (cells per mL)		Inhibition Values (%)
Nominal Concentration (mg/L)		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.77E+03	5.41E+05
	R2	6.46E+03	6.27E+05	-	-
	Mean	6.62E+03	5.84E+05
0.10	R1	6.69E+03	6.37E+05
	R2	5.66E+03	5.83E+05	[3]	[5]
	Mean	6.18E+03	6.10E+05
1.0	R1	6.72E+03	7.13E+05
	R2	5.14E+03	3.28E+05	0	11
	Mean	5.93E+03	5.20E+05
10	R1	5.76E+03	7.22E+04
	R2	5.71E+03	7.33E+04	44	88
	Mean	5.74E+03	7.28E+04
100	R1	6.32E+03	9.33E+03
	R2	5.81E+03	8.24E+03	92	100

Table 2. Cell density and pH values - Definitive test

Nominal Concentration (mg/L)		pH	Cell Densities (cells per mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.5	5.15E+03	2.20E+04	1.14E+05	6.32E+05	8.0
	R2		5.17E+03	2.45E+04	1.65E+05	9.74E+05
	R3		5.01E+03	2.90E+04	1.29E+05	8.75E+05
	R4		4.61E+03	2.47E+04	1.28E+05	8.03E+05
	R5		4.94E+03	2.77E+04	1.56E+05	8.31E+05
	R6		4.70E+03	2.39E+04	1.55E+05	8.50E+05
	Mean		4.93E+03	2.53E+04	1.41E+05	8.27E+05
1.0	R1	7.4	4.75E+03	2.64E+04	1.63E+05	7.66E+05	7.9
	R2		4.91E+03	3.01E+04	1.30E+05	9.24E+05
	R3		4.99E+03	2.80E+04	1.38E+05	6.35E+05
	Mean		4.89E+03	2.82E+04	1.44E+05	7.75E+05
3.2	R1	7.4	5.39E+03	3.07E+04	1.87E+05	9.09E+05	7.9
	R2		4.75E+03	2.81E+04	1.70E+05	1.06E+06
	R3		5.25E+03	2.89E+04	1.14E+05	6.95E+05
	Mean		5.13E+03	2.92E+04	1.57E+05	8.87E+05
10	R1	7.3	5.74E+03	2.10E+04	5.62E+04	1.00E+05	7.8
	R2		4.91E+03	2.32E+04	5.01E+04	8.32E+04
	R3		4.64E+03	1.72E+04	4.43E+04	9.03E+04
	Mean		5.10E+03	2.05E+04	5.02E+04	9.12E+04
32	R1	7.3	4.94E+03	1.47E+04	1.49E+04	1.93E+04	7.7
	R2		5.39E+03	1.30E+04	1.51E+04	2.24E+04
	R3		5.89E+03	1.26E+04	1.52E+04	2.25E+04
	Mean		5.41E+03	1.35E+04	1.51E+04	2.14E+04
100	R1	7.2	6.02E+03	7.18E+03	9.94E+03	9.10E+03	7.6
	R2		4.91E+03	6.85E+03	8.45E+03	9.70E+03
	R3		4.74E+03	7.50E+03	8.85E+03	8.24E+03
	Mean		5.22E+03	7.18E+03	9.08E+03	9.01E+03

Table 3. Daily specific growth rates for the control cultures - Definitive test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.062	0.068	0.071
	R2	0.066	0.079	0.074
	R3	0.073	0.062	0.080
	R4	0.067	0.069	0.076
	R5	0.071	0.072	0.070
	R6	0.065	0.078	0.071
	Mean	0.067	0.071	0.074

Table 4. Inhibition of growth rate and yield - Definitive test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 - 72h	% Inhibition	0 - 72h	%Inhibition
Control	R1	0.067		6.27E+05
	R2	0.073		9.69E+05
	R3	0.072		8.70E+05
	R4	0.071	-	7.99E+05	-
	R5	0.071		8.26E+05
	R6	0.071		8.46E+05
	Mean	0.071		8.22E+05
	SD	0.002		1.12E+05
1.0	R1	0.070	1	7.61E+05
	R2	0.072	[1]	9.19E+05
	R3	0.067	6	6.30E+05
	Mean	0.070	2	7.70E+05	6
	SD	0.003		1.45E+05
3.2	R1	0.072	[1]	9.03E+05
	R2	0.074	[4]	1.05E+06
	R3	0.069	3	6.90E+05
	Mean	0.072	[1]	8.82E+05	[7]
	SD	0.003		1.82E+05
10	R1	0.042	41	9.44E+04
	R2	0.039	45	7.82E+04
	R3	0.040	44	8.56E+04
	Mean	0.040	43	8.61E+04	90
	SD	0.002		8.09E+03
32	R1	0.019	73	1.44E+04
	R2	0.021	70	1.70E+04
	R3	0.021	70	1.66E+04
	Mean	0.020	71	1.60E+04	98
	SD	0.001		1.41E+03
100	R1	0.008	89	3.09E+03
	R2	0.009	87	4.78E+03
	R3	0.007	90	3.50E+03
	Mean	0.008	89	3.79E+03	100
	SD	0.001		8.84E+02

Validity criteria fulfilled:

yes

Remarks:

All validity criteria of the applied test guideline were met.

Conclusions:

The report describes a valid guideline study conducted under certificated GLP compliance. The obtained 72h-ErC50 value (15 mg/L) leads to a certain toxicity concern of the test substance towards freshwater algae.

Executive summary:

The toxicity of Diphenyl methylphosphonate (DPP) towards freshwater algae was investigated according to OECD Guideline 201 / EU Method C.3 using Pseudokirchneriella subcapitata as test organism (Vryenhoef, 2013). Pseudokirchneriella subcapitata is an important non-target organism in freshwater ecosystems since it is a representative of primary producers found in natural waters. After a preliminary range-finding test, the substance concentrations for the definitive test were chosen as: 1.0, 3.2, 10, 32 and 100 mg/L (3 replicates per concentration). The test substance was heated to 50 °C (in order to aid weighing) and afterwards dissolved in culture medium with the aid of ultrasonication. The concentration and stability of the test substance in the preparations as well as the pH values were verified by chemical analysis at the beginning and termination of the experiment. The temperature within the incubator was recorded daily. Analytical investigations were performed using High Performance Liquid Chromatography with UV detection (HPLC-UV) using an external standard. The validity of the analytical procedure was confirmed by spiked recovery samples (test medium were spiked with the test substance and recovery was assessed). The initial cell density was 5 x 10E3 cells/mL. The test flasks were plugged with polyurethane foam bungs and incubated at 24 +/- 1 °C under continuous illumination (approx. 7000 lux) and constantly shaken at approx. 150 rpm for the exposure period of 72 hours. A control group (six replicates) was maintained under identical conditions but not exposed to the test substance. Samples of the algal population were removed daily and cell concentrations were determined for each culture group using a Coulter® Multisizer Particle Counter. Potassium dichromate served as reference substance, giving the following results: ErC50(72h): 1.2 mg/L (95 % CL: 1.0 – 1.3 mg/L) and EbC50(72h): 0.52 mg/L (95 % CL: 0.45 – 0.62 mg/L) with corresponding NOECs of 0.25 mg/L and LOECs of 0.50 mg/L. These results were within the normal ranges. All cultures were inspected microscopically after the exposure period. No abnormalities were detected in the control and test cultures of 1.0 and 3.2 mg/L, however few intact cells and cell debris was observed at 10 and 32 mg/L. Almost no intact cells were observed in the 100 mg/L cultures. All cultures were observed to be clear colorless solutions at the start of the experiment. The controls as well as the 1.0 mg/L and 3.2 mg/L cultures were observed to be green dispersions after 72 hours. Statistical analysis was carried out using 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 (Dunnet, 1955). Based on the inhibition of growth rate, the following results were reported: ErC10(72h): 3.1 mg/L, ErC20(72h): 5.6 mg/L and ErC50(72h): 15 mg/L (with 95 % CL: 12 – 19 mg/L). Taken into account the inhibition of yield, the results are as followed: EyC10(72h): 7.1 mg/L, EyC20(72h): 7.5 mg/L and EyC50(72h): 8.4 mg/L (these data did not fit the models available for the calculation of confidence limits). The corresponding No Observed Effect Concentrations (NOEC) is 3.2 mg/L for each method of approach. The Lowest Observed Effect Concentrations (LOEC) was found as 10 mg/L. All validation criteria of the applied guideline were met.

Description of key information

Pseudokirchneriella subcapitata_OECD 201/EU Method C.3: ErC10(72h): 3.1 mg/L, ErC20(72h): 5.6 mg/L, ErC50(72h): 15 mg/L / EyC10(72h): 7.1 mg/L, EyC20(72h): 7.5 mg/L, EyC50(72h): 8.4 mg/L / NOEC(72h): 3.2 mg/L / LOEC(72h): 10 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

15 mg/L

EC10 or NOEC for freshwater algae:

3.1 mg/L

Additional information

The toxicity of Diphenyl methylphosphonate (DPP) towards freshwater algae was investigated according to OECD Guideline 201 / EU Method C.3 using Pseudokirchneriella subcapitata as test organism (Vryenhoef, 2013). Pseudokirchneriella subcapitata is an important non-target organism in freshwater ecosystems since it is a representative of primary producers found in natural waters. After a preliminary range-finding test, the substance concentrations for the definitive test were chosen as: 1.0, 3.2, 10, 32 and 100 mg/L (3 replicates per concentration). The test substance was heated to 50 °C (in order to aid weighing) and afterwards dissolved in culture medium with the aid of ultrasonication. The concentration and stability of the test substance in the preparations as well as the pH values were verified by chemical analysis at the beginning and termination of the experiment. The temperature within the incubator was recorded daily. Analytical investigations were performed using High Performance Liquid Chromatography with UV detection (HPLC-UV) using an external standard.The validity of the analytical procedure was confirmed by spiked recovery samples (test medium were spiked with the test substance and recovery was assessed). The initial cell density was 5 x 10E3 cells/mL. The test flasks were plugged with polyurethane foam bungs and incubated at 24 +/- 1 °C under continuous illumination (approx. 7000 lux) and constantly shaken at approx. 150 rpm for the exposure period of 72 hours. A control group (six replicates) was maintained under identical conditions but not exposed to the test substance. Samples of the algal population were removed daily and cell concentrations were determined for each culture group using a Coulter® Multisizer Particle Counter. Potassium dichromate served as reference substance, giving the following results: ErC50(72h): 1.2 mg/L (95 % CL: 1.0 – 1.3 mg/L) and EbC50(72h): 0.52 mg/L (95 % CL: 0.45 – 0.62 mg/L) with corresponding NOECs of 0.25 mg/L and LOECs of 0.50 mg/L. These results were within the normal ranges. All cultures were inspected microscopically after the exposure period. No abnormalities were detected in the control and test cultures of 1.0 and 3.2 mg/L, however few intact cells and cell debris was observed at 10 and 32 mg/L. Almost no intact cells were observed in the 100 mg/L cultures. All cultures were observed to be clear colorless solutions at the start of the experiment. The controls as well as the 1.0 mg/L and 3.2 mg/L cultures were observed to be green dispersions after 72 hours. Statistical analysis was carried out using 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 (Dunnet, 1955). Based on the inhibition of growth rate, the following results were reported: ErC10(72h): 3.1 mg/L, ErC20(72h): 5.6 mg/L and ErC50(72h): 15 mg/L (with 95 % CL: 12 – 19 mg/L). Taken into account the inhibition of yield, the results are as followed: EyC10(72h): 7.1 mg/L, EyC20(72h): 7.5 mg/L and EyC50(72h): 8.4 mg/L (these data did not fit the models available for the calculation of confidence limits). The corresponding No Observed Effect Concentrations (NOEC) is 3.2 mg/L for each method of approach. The Lowest Observed Effect Concentrations (LOEC) was found as 10 mg/L. All validation criteria of the applied guideline were met.