Reaction mass of dimethyl phosphonate and methyl hydrogenphosphonate and phosphonic acid

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:

other information

Reliability:

4 (not assignable)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

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

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Executive summary:

For the parameter growth rate , the 72-hour ErC50 value was calculated to be 153 mg test item/L and the 72h-NOEC was 1.0 mg/L

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

other information

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

cited as Directive 92/69/EEC, C.3; in most parts equivalent to the OECD TG 201

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- samples were taken at the start (0 hrs) and end (72 hrs) of the experiment
- samples from control and test concentrations were analysed

Vehicle:

no

Details on test solutions:

To produce the only test concentration 125.1 mg of the test item were added to 1 litre of dilution water and treated for 30 minutes on a magnetic stirrer.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): non-axenic strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany)

MAINTENANCE OF STOCK CULTURES
Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions (23 +/- 2°C) at a light intensity in the range 60 - 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to Bringmann and Kuehn (1977) is renewed once a week. Cell density measurements are made using a microcell counter.

PREPARATION OF PRE-CULTURES
Pre-cultures are set up three days before the start of a test. They are grown under identical exposure conditions as the stock cultures, except from the use of a different nutrient medium (see below).

TEST CULTURES
The algal inocula for a test are taken from an exponentially-growing pre-culture and are mixed with the nutrient medium (see below) to make up to a final cell density of about 10E4 cells per millilitre in the test medium.

NUTRIENT MEDIUM OF PRE-CULTURES AND TEST CULTURES
=> see "details on test conditions"

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21-25°C ± 2°C

pH:

start of the experiment (0 h): 8.2 (control) and 8.1 (100 mg/L)
end of experiment (72 h): 10.5 (control) and 10.6 (100 mg/L)

Nominal and measured concentrations:

nominal concentration: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 ml Erlenmeyer flasks with stoppers
- Type: closed
- Initial cells density: 10E4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM OF PRE-CULTURES AND TEST CULTURES
- the nutrient medium is obtained from the following 3 stock solutions:

Stock solution 1: macro-nutrients
NH4Cl: 15 mg/l
MgCl2 x 6 H2O: 12 mg/l
CaCl2 x 2 H2O : 18 mg/l
MgSO4 x 7 H2O: 15 mg/l
KH2PO4: 1.6 mg/l

Stock solution 2: Fe-EDTA
FeCl3 x 6 H2O: 80 µg/l
Na2EDTA x 2 H2O: 100 µg/l

Stock solution 3: trace elements
H3BO3: 185 µg/l
MnCl2 x 4 H2O: 415 µg/l
ZnCl2: 3 µg/l
CoCl2 x 6 H2O: 1.5 µg/l
CuCl2 x 2 H2O: 0.01 µg/l
Na2MoO4 x 2 H2O: 7 µg/l

Solid NaHCO3 is added to the nutrient media to make up a final concentration of 50 mg/l in the solutions of the pre-cultures and test cultures.

OTHER TEST CONDITIONS
- Light intensity and quality: 60-120 µE x m² x s-1
- Light spectral range: 400-700 nm

EFFECT PARAMETERS MEASURED:
- Effect parameters: growth and growth rate
- Determination of cell concentrations: by microcell counter or a microscopic counting chamber
- Observation intervals: 24 hour intervals (24 h, 48 h, 72 h)

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Inhibition of algal growth - summary of raw data

table 1: mean cell density during the test

Nominal concentration [mg/l]	Cell density [cells/ml]
	24 h	48 h	72 h
Control	50000	146111	333333
100	60000	186667	432222

 

table 2: mean growth [integral of biomass]

Nominal concentration [mg/l]	Area under growth curve	Inhibition (+) / Increase (-) [%]
Control	337778	0.0
100	437778	-29.6

 

table 3: mean growth rate

Nominal concentration [mg/l]	Growth rate [1/d]	Inhibition (+) / Increase (-) [%]
Control	1.17	0.0
100	1.26	-7.4

 

 

Chemical analysis:

The results of two different analytical methods (HPLC and DOC-determination) clearly demonstrate that dimethyl phosphonate hydrolysed within a few hours and that the overall concentration of organic carbon (measured by DOC) was stable throughout the complete exposure period. Therefore the results were expected based on nominal concentrations.

table 4: chemical analysis

	control		100 mg/l
	0 h	72 h	0 h	72 h
DOC replicates [mg/l]	< 5 / < 5	10 / 10	22 / 21	22 / 22
DOC mean value [mg/l]	< 5	10	21.5	22.0
HPLC replicates Dimethylphosphit [mg/l]	< 0.375	< 0.375	< 0.375	< 0.375
HPLC replicates Monomethylphosphit [mg/l]	< 0.375*	< 0.375*	25.28*	26.96*

* Values calculated as dimethylphosphit.

Description of key information

For phosphonic ester residue, there is no study on algae or cyanobacteria available. However, for two components, dimethyl phosphonate (CAS 868-85-9, DMP) and phosphonic acid (13598-36-2, PA) such studies are available. In the study with DMP, dimethyl phosphonate was hydrolysed completely to monomethyl phosphonate (CAS 13590-71-1, MMP) within about 0.5 h during the preparation of the test solutions. MMP remained stable during the test for 72 h. Thus, it is plausible to assume that the study with DMP considers also potential effects of MMP, the third component of the registered substance.

In the study with DMP, no toxic effect on growth rate of Desmodesmus subspicatus could be observed, thus the 72 h-EC50 (nominal) was = 100 mg/L. 

In the study with PA, the 72-hour ErC50 value was calculated to be 153 mg test item/L.

Thus, one can assume, that phosphonic ester residue shows no effects up to 100 mg/L.