Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium 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:

2009-04-07 until 2009-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, 2008-11-12

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/l of dry salt of test item cC604
- Sampling method: For measurement of the actual concentrations of the test item cC6O4, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test medium of the highest nominal test item concentration of 625 mg cC6O4/L was prepared by mixing 312.5 mg of the test item cC6O4 into 500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. This test medium was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the test.
- Method of cultivation: The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.


ACCLIMATION
Not applicable. The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no data

Hardness:

0.24 mmol/L as CaCO3

Test temperature:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C

pH:

At the start of the test, the pH measured in the treatments was 8.5. At the end of the test, pH values of 8.2 to 8.3 were measured

Dissolved oxygen:

no data; during the test, the test solutions were continuously stirred by magnetic stirrers.

Salinity:

No data, however detailed description of the mineral composition of the test medium is given in the study report.

Nominal and measured concentrations:

The following nominal concentrations of the test item cC6O4 were tested: 6.25, 20, 62.5, 200 and 625 mg cC6O4/L, corresponding to 1.0, 3.2, 10, 32 and 100 mg dry salt cC6O4/L (test item cC6O4 contains 84% water).
The measured concentrations of the test item cC6O4 (calculated based on the measurement of the content of dry salt cC6O4) in the test media of the highest test item concentration of cC6O4 were between 103 and 106% of the nominal values at the start and the end of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 50-mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution.
- Aeration: During the test period, the test water was not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): no renewal
- No. of organisms per vessel: The test was started using a nominal algal cell density of 10000 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: The biomass was determined by fluorescence measurement and is given as relative fluorescence units (x 10^3). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 1.72 x 10^3 relative fluorescence units.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing.
- Total organic carbon: no data
- Particulate matter: no data
- Metals: Macro nutrients: Na, K, Ca, Mg; Micro nutrients: B, Mn, Zn, Co, Cu, Mo, Fe (plus EDTA)
- Pesticides: not applicable
- Chlorine: not applicable
- Alkalinity: 0.8 mmol/l
- Ca/Mg ratio: 1:6.3 (mol/mol)
- Conductivity: No data, however detailed description of the mineral composition of the test medium is given in the study report.
- Culture medium different from test medium: no
- Intervals of water quality measurement: samples taken at the start and at the end of the test after 72 hours.


OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: Flasks were illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily.
- Light intensity: The mean measured
light intensity at the level of the test solutions was approximately 7800 Lux (range: 7200 to 8400 Lux, measured at nine places in the experimental area).


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density was measured via Coulter counter; biomass was measured via fluorometric method.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: The enlarged spacing factor of 3.2 between the test concentrations was chosen, because according to the results of the range-finding test the concentration-effect relationship was rather flat and, thus, a large concentration range had to be tested.
- Range finding study: The selection of the test concentrations was based on the results of a range-finding test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: none reported

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in February 2009 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.1 mg/L (study C36893), range of the 72-hour EC50 for the growth rate from 2000 to 2009: 0.71-1.74 mg/L).

Reported statistics and error estimates:

Dunnett’s tests, one-sided, α = 0.05

Tab. 1: Average Growth Rates (μ)

Dry salt cC604 (mg/l)	Average growth rate μ (day-1) and inhibition of μ (Ir)
	0-24 h		0-48 h		0 -72 h
	µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	1.61	0	1.58	0	1.4	0
1.0	1.68	-4.4	1.61	-2.1	1.39	0.4
3.2	1.59	0.9	1.61	-2.1	1.45	-3.7
10	1.53	4.6	1.57	0.9	1.35	3
32	1.53	5.1	1.56	1.4	1.34	4.1
100	1.47	8.6	1.59	-0.5	1.39	0.6

Tab. 2: Average Growth Rates (μ)

Dry salt cC604 (mg/l)	Yield Y (x 103) and inhibition of Y (Iy)
	0-24 h		0-48 h		0 -72 h
	Y	Iy (%)	Y	Iy (%)	Y	Iy (%)
Control	6.9	0	39	0	113.7	0
1.0	7.5	-8.9	41.7	-7.1	110.8	2.6
3.2	6.8	1.8	41.7	-7.0	131.8	-15.9
10	6.3	8.5	37.8	2.9	99	12.9
32	6.2	9.9	37.5	3.7	95.2	16.2
100	5.8	16.3	39.6	-1.6	110.1	3.2

Validity criteria fulfilled:

yes

Remarks:

negative and positive controls valid

Conclusions:

The test item cC6O4 ammonium salt had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the highest nominal dry salt concentration of 100 mg cC6O4 ammonium salt/L.

Executive summary:

The influence of the test item cC6O4 ammonium salt on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006), the EU Commission Directive 92/69/EEC, C.3 (1992) and the Commission Regulation (EC) No 440/2008, C.3. The nominal concentrations of the test item of 6.25, 20, 62.5, 200 and 625 mg cC6O4 ammonium salt/L, corresponding to 1.0, 3.2, 10, 32 and 100 mg dry salt cC6O4 ammonium salt/L (test item cC6O4 ammonium salt contains 84% water) were tested in parallel with a control. The test item cC6O4 ammonium salt had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the highest nominal dry salt cC6O4 ammonium salt concentration of 100 mg/L. The nominal dry salt cC6O4 ammonium salt concentration of 100 mg/L was, therefore, determined to be the 72-hour NOEC. This value might even be higher, but nominal concentrations of dry salt cC6O4 ammonium salt exceeding 100 mg/L were not tested, in accordance with the test guidelines. The 72-hour LOEC was higher than 100 mg dry salt cC6O4 ammonium salt/L.

Description of key information

The influence of the test item cC6O4 ammonium salt on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was determined in a 72-hour static test according to the OECD Guideline 201 (2006), the EU Commission Directive 92/69/EEC, C.3 (1992) and the Commission Regulation (EC) No 440/2008, C.3.
The study was performed under GLP without deviations.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

No adverse effects were observed up to the nominal concentration of 625 mg test item cC6O4 ammonium salt/L corresponding to 100 mg dry salt cC6O4 ammonium salt/L (test item = aqueous solution of cC6O4 ammonium salt containing 84% water).

Nominal concentrations of dry salt cC6O4 ammonium salt exceeding 100 mg/L were not tested, in accordance with the test guidelines.

The 72-hour Ec50 was higher than 100 mg dry salt cC6O4 ammonium salt/L.