Phosphonic acid, monoethyl ester, sodium salt (1:1)

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:

28 May - 01 Jun 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency, Pesticide Assessment Guidelines, Subdivision J, Hazard Evaluation: Nontarget Plants §123-2: Growth and reproduction of aquatic plants (Tier 2), (EPA 540/9-82-020 from 27. October 1982).

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Jugend, Familie und Gesundheit

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of the freshly prepared and aged (96h) test solutions was performed for all tested concentrations. The concentrations were
diluted prior analyses.

Vehicle:

no

Details on test solutions:

Calculations of the aqueous concentrations of test item were based on 22.1% purity of the test material. To obtain fresh stock solution for the test, the test item was weighed out to a precision of 0.1 mg and dissolved in nutrient medium. Each test solution was prepared individually. Defined amounts of the stock solution, well shaken before extraction, were pipetted proportionally. Defined amounts of pre-culture were added into each of the corresponding graduated cylinder to obtain a final cell concentration of 10^4 cells/mL and then filled up to the exactly volume with nutrient medium. For each concentration at hree-fold amount was prepared for the replicates treated with test item. The residual amount was used for test item analysis or discarded.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: 61.81
- Source (laboratory, culture collection): Collection of Algal Cultures, Institute of Plant Physiology, University of Goettingen, D-37073 Goettingen, FRG
- Pre-culture: Four days prior to testing a pre-culture was started in the nutrient medium: 300mL Erlenmeyer flasks closed with pressed paper stoppers, 100 mL freshly prepared nutrient medium/flask, a cell concentration of 10^4 cells/mL at the start. Test culture conditions were identical to pre-culture conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

0.22 total hardness in mmol/L (Ca2+ & Mg2+)

Test temperature:

24.1 - 25.2 °C

pH:

7.0 - 9.6

Dissolved oxygen:

7.5 - 8.6 mg O2/L

Nominal and measured concentrations:

control, 10, 18, 32, 56, and 100 mg/L (active ingredient: AE C529354, nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with pressed paper stoppers
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks, which were filled with 100mL medium
- Initial cells density: 10000 cells/mL
- Control end cells density: 32 x 10^4 (72 h), 106.4 x 10^4 (96 h)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes. according to OECD and EPA guidelines

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reagent grade water is prepared from deionized water - obtainable throughout the facility - which is additionally filtered by an ultrafiltration, ion exchange and a charcoal unit.
- Conductivity: 105 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: The analyses of conductivity, total hardness, and acid binding capacity were made during the first test day. Oxygen concentration and pH values of each test flask and temperature at each concentration step were assessed daily.

OTHER TEST CONDITIONS
- Adjustment of pH: yes, to pH 7.5 at experimental start
- Photoperiod: continuous
- Light intensity and quality: wides pectrum fluorescent lamps of the universal white-type and a mean light intensity of about 66 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Samples of the algal populations were removed daily from each test vessel and cell concentrations were determined after 24, 48, 72 and 96 hours test duration. Algal morphology was also observed daily using a light microscope and countingchamber.
- Determination of cell concentrations: electronic particle counter - Coulter Multisizer II

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: equivalent to EC50 >452 mg AEC529354 technical concentrate 22.1%/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: equivalent to EC50 >452 mg AEC529354 technical concentrate 22.1%/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: equivalent to EC50 >452 mg AEC529354 technical concentrate 22.1%/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: equivalent to EC50 >452 mg AEC529354 technical concentrate 22.1%/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

and cell morphology

Remarks on result:

other: equivalent to NOEC = 452 mg AEC529354 technical concentrate 22.1%/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

and cell morphology

Remarks on result:

other: equivalent to NOEC = 452 mg AEC529354 technical concentrate 22.1%/L

Details on results:

No treatment level showed any significant growth inhibition.

Chemical analysis:

Analyses of freshly prepared water for AE C529354 resulted in test item concentrations ranging from 93.5% to 104.3% of nominal values. Analyses of aged water (96h) for AE C529354 at experimental termination resulted in test item concentrations ranging from 97.3% to 117.5% of nominal values. The mean measured values over the time of exposure ranged from 96.2% to 110.9%. Since all analysed concentrations of AE C529354 were within ± 20% of nominal at the start and the end of the study, all effect concentrations were based on nominal initial test concentrations.

Table 1: Cell numbers - Mean values

Nominal treatment level (mg/L)	Mean cell numbers x 10^4 after …
	24 h	48 h	72 h	96 h
Control	2.6	7.1	32.0	106.4
10	2.4	9.2	41.9	142.3
18	2.7	8.0	37.3	115.0
32	2.5	9.0	38.5	121.3
56	2.2	7.9	35.9	110.5
100	2.0	8.4	36.9	105.7

Table 2: Areas under the growth curves, growth rates - Mean values after 72 hours

Nominal treatment level (mg/L)	Mean area under the growth curve (10^4/mL*h) after 72 h	Mean growth rate/h after 72 h	Percentual inhibition according to mean area under the growth curve after 72 h	Percentual inhibition according to mean growth rate after 72 h
Control	555.66	0.04804	0.00	0.00
10	721.68	0.05184	-29.88	-7.90
18	643.56	0.05027	-15.82	-4.64
32	678.20	0.05067	-22.05	-5.46
56	613.32	0.04968	-10.38	-3.40
100	633.32	0.05011	-13.98	-4.29

Table 3: Areas under the growth curves, growth rates - Mean values after 96 hours

Nominal treatment level (mg/L)	Mean area under the growth curve (10^4/mL*h) after 96 h	Mean growth rate/h after 96 h	Percentual inhibition according to mean area under the growth curve after 96 h	Percentual inhibition according to mean growth rate after 96 h
Control	2191.32	0.04852	0.00	0.00
10	2907.24	0.05164	-32.67	-6.42
18	2447.40	0.04942	-11.69	-1.86
32	2571.68	0.04996	-17.36	-2.96
56	2345.76	0.04899	-7.05	-0.96
100	2320.24	0.04852	-5.88	-0.00

Table 4: Validity criteria OECD 201

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The biomass increased with a factor of 32 at 72 h and a factor of 106 at 96 h	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation for section-by-section specific growth rates was 27% at 72 h and 23% at 96 h	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	The coefficient of variation of average specific growth rates was 3.2% at 72 and 96 h	Yes

Validity criteria fulfilled:

yes

Remarks:

see Table 4 in 'any other information on results incl. tables'

Description of key information

EbC50 and ErC50 (72 h) > 100 mg a.i. (Fosetyl-Sodium)/L for Pseudokirchneriella subcapitata (OECD 201) (equivalent to > 452 mg Fosetyl-Sodium technical concentrate 22.1%/L) 
NOEC (72 h) = 100 mg (Fosetyl-Sodium)/L for Pseudokirchneriella subcapitata (OECD 201) (equivalent to 452 mg Fosetyl-Sodium technical concentrate 22.1%/L)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A GLP study according to OECD 201 on growth inhibition of the green alga Pseudokirchneriella subcapitata was performed with Fosetyl-sodium (technical concentrate; 22.1%; Code: AE C529354 00 TK22 A101; containing 22.1% w/w Fosetyl-Sodium) under static conditions.

Triplicate algal cultures with an initial cell density of 10 000 algal cells/mL were incubated in a synthetic medium at 25 ± 1 °C for 96 hours. Nominal test item concentrations were 10, 18, 32, 56 and 100 mg Fosetyl-Sodium/L with three replicates each, together with an untreated control with six replicates. The low purity of the technical concentrate was taken into consideration. Samples of the algal populations were removed daily from each test vessel and cell concentrations were determined after 24, 48, 72 and 96 hours test duration. Algal morphology was also observed daily.

Chemical analysis of the freshly prepared and aged (96 h) test solutions was performed for all tested concentrations of Fosetyl-Sodium using High Performance Liquid Chromatography with ultraviolet detection (HPLC-UV). The concentrations were diluted prior analyses. Analyses of freshly prepared water for Fosetyl-Sodium resulted in test item concentrations ranging from 93.5% to 104.3% of nominal values. Analyses of aged water (96 h) for the test item at experimental termination resulted in concentrations ranging from 97.3% to 117.5% of nominal values. The mean measured values over the time of exposure ranged from 96.2% to 110.9%. Since all analyzed concentrations of the test item were within ± 20% of nominal at the start and the end of the study, all effect concentrations were based on nominal initial test concentrations. No treatment level showed any significant growth inhibition.

The nominal concentration of test substance inhibiting the growth and the resulting EbC50 (concentration for a 50% reduction of growth based on a comparison of areas under the growth curves) in comparison with the untreated control after 72 and 96 hours test duration was >100 mg test item /L (equivalent to > 452 mg technical concentrate 22.1% / L). The nominal concentration of test substance inhibiting the growth and the resulting ErC50 (concentration for a 50% reduction of specific growth rate based on a comparison of slopes of the growth curves) in comparison with the untreated control after 72 and 96 hours test duration was >100 mg test item/L (equivalent to > 452 mg technical concentrate 22.1% / L).

No significant inhibition of growth based on a comparison of areas under the growth curves (significance level of alpha = 0.05) was observed in any of the tested concentrations. No significant inhibition of specific growth rate based on a comparison of slopes of the growth curves (significance level of alpha = 0.05) was observed in any of the tested concentrations after 72 and 96 hours test duration. Therefore, the no observed effect concentration (NOEC), defined as the concentration which had no significant effect on growth inhibition or cell morphology, after 72 and 96 hours was 100 mg/L, the highest tested concentration.