Phosphorothioic acid, O,O-bis(2-methylpropyl) ester, sodium salt (1:1)

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:

19 - 22 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium O,O-diisobutyl phosphorothioate

EC Number:

610-992-2

Cas Number:

53378-52-2

Molecular formula:

C8 H18 O3 P S . Na

IUPAC Name:

Sodium O,O-diisobutyl phosphorothioate

Test material form:

other: grease-like solid

Specific details on test material used for the study:

- Name of test material (as cited in study report): S10783
- Name: Sodium diisobutyl monothiophoshate
- CAS: 53378-52-2
- Molecular formula: C8H18O3PS.Na
- Molecular weight: 248.26
- Storage condition of test material: not indicated in the study report
- Purity: 70%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Control and 100 mg/L (with and without algae)
- Sampling method:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 1 ml

- Sample storage conditions before analysis: Samples were stored in a freezer until analysis

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Preparation of test solutions started with a loading rate of 100 mg/L applying 10 minutes of magnetic
stirring to dissolve the test substance in the test medium. The lower test concentrations were prepared
by subsequent dilutions of the 100 mg/L concentration in test medium. The final test solutions were all
clear and colourless.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/ml.
- Culturing media and conditions (same as test or not): Pre-culture medium same as test medium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L as Ca+Mg (24 mg CaCO3/L)

Test temperature:

22.3 and 23.7°C

pH:

8.0-8.1

Nominal and measured concentrations:

- Nominal conc. (mg/L)
Control, 0.10, 1, 10, 100

- Measured conc. (mg/L)
at 0 h: Control = not detected; 100 (nominal) = 93.9
at 24 h: Control = not detected; 100 (nominal) = 93.6
at 72 h: Control = small response was detected at the test substance retention time. The contribution based on area was 0.004% to the 100 mg/l samples.; 100 (nominal) = 93.2

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessels
- Type: closed
- Material, size, headspace, fill volume: 100 ml, all-glass, containing 50 ml of test solution
- Aeration: None
- Initial cells density: 1x 10^4 cells/ml.
- No. of vessels per concentration (replicates): 3 replicates for lower concentrations and 6 replicates for highest conc.
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water (tap water purified by reverse osmosis (Milli-
RO) and subsequently passed over activated carbon and
ion-exchange cartridges: Milli-Q water; Millipore Corp.,
Bedford, Mass., USA)
- Culture medium different from test medium: Pre-culture medium same as test medium
- Intervals of water quality measurement:
pH measured at the beginning and at the end of the test.
Temperature was continuously measured in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: 60 to 120 μE/m2/s when measured in the photosynthetically
effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
At the beginning of the test, cells were counted using a microscope and a counting chamber.
Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm
using a spectrophotometer with immersion probe (pathlength =20 mm).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations:
Control, 0.10, 1.0, 10 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

At the start of the test, the actual test concentration at nominal 100 mg/l was in agreement with nominal (94%) and this concentration remained stable during the test period (99% of initial at the end of the test). Given this result, the effect parameters were based on nominal exposure concentrations.

Results with reference substance (positive control):

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations 0.32 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 0.98 mg/l with a 95% confidence interval ranging from 0.74 to 1.3 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.44 mg/l with a 95% confidence interval ranging from 0.32 to 0.61 mg/l. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Any other information on results incl. tables

Percentage reduction of growth rate (total test period) and percentage inhibition of yield

Nominal conc.	Mean growth rate		Yield (0-72 h)
S-10783
(mg/l)	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
control	0.07330		195.06
0.10	0.07366	-0.5	200.18	-2.6
1.0	0.07308	0.3	191.82	1.7
10	0.07299	0.4	190.52	2.3
100	0.07244	1.2	183.42	6.0

Concentrations of the test substance in test medium
			Concentration
Time of sampling [hours]	Date of sampling [dd-mm-yy]	Date of analysis^1[dd-mm-yy]	Nominal [mg/l]	Analysed [mg/l]	Relative to nominal [%]	Relative to initial [%]
0	19/06/2012	26/06/2012	0	nd	na
			100	93.9	94
			100^2	102	102
24	20/06/2012	26/06/2012	0	nd	na	na
			100	93.6	94	100
			100^2	93.3	93	92
72	22/06/2012	26/06/2012	0	3	na	na
			100		93	99
			100^2		86	84

1) Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2) Without algae.

3) A small response was detected at the test substance retention time. The contribution based on area was 0.004% to the

100 mg/l samples. The result of the reserve sample was similar.

n.d.) Not detected.

n.a.) Not applicable.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations tested. The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) was >100 mg/L.

Executive summary:

The toxicity of the test material to aquatic algae and cyanobacteria was assessed according to OECD TG 201 and GLP principles. Nominal exposure concentrations were the following: control, 0.10, 1.0, 10, 100 mg/L.

The test material did not reduce the growth rate or inhibited the yield of the fresh water algae speciesPseudokirchneriella subcapitataat a nominal exposure concentration of 100 mg/L. The EC50 for growth rate reduction (ErC50: 0-72h) was >100 mg/l with a NOEC of 100 mg/L. For the yield inhibition, the EC50 (EyC50: 0-72h) was >100 mg/L with a NOEC 100 mg/L.

All criteria for acceptability of the test were met and the present toxicity study is classified as reliable without restrictions according to the OECD TG 201.