Propanoic acid, 2,2,3-trifluoro-3-[1,1,2,2,3,3-hexafluoro-3-(trifluoromethoxy)propoxy]-, methyl ester

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:

14 July 2009 - 16 July 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP, test substance could not be maintained in solution, all reasonable steps taken to maintain test substance concentration

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

other: OECD Series on Testing and Assessment No. 23

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

MeDONA

IUPAC Name:

MeDONA

Details on test material:

- Name of test material (as cited in study report): MTDID #10078
- Physical state: clear colorless liquid
- Analytical purity: 91.9%
- Lot/batch no.: 140499-21/8
- Stability under test conditions: Hydrolytically unstable and likely to be volatile
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At 0h and 24h. 4.5 mL aliquots were taken from all test concentration and controls.
- Sample storage conditions before analysis: Not applicable, samples were analysed same day as sampling because MeDONA concentration were not stable during freezing and thawing of samples.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest concentration (loading rate of 100 mg/L) was prepared by spiking underneath the surface a volume of 63 µL test substance to 1L test medium. The aqueous solution was magnetically stirred for 21 hours in a closed glass vessel with minimal headspace in order to prevent any volatilization and to reach maximum solubility. The mixture was then stabilized for one hour and then the water accommodated fraction (WAF) was siphoned off. The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the WAF in test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material: The final test solutions were all clear and colorless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algae cells from a pure culture on agar. Suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 - 24°C

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Combined limit/range-finding test

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

22.2 - 23.0 °C

pH:

7.4 - 9.1

Nominal and measured concentrations:

Water accommodated fraction: 0.1, 1.0, 10 and 100% from a loading rate of 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:100 mL all-glass, containing 75 mL of test solution. The vessels were airtight closed with a screw-cap that contained a septum.
- Agitation: Algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 23.85E+04 cells/mL
- No. of vessels per concentration (replicates): 3 for 0.1, 1.0 and 10% WAF. 6 for 100% WAF.
- No. of vessels per control (replicates): 6
1 or 2 replicates of each test concentration for analytical sampling purposes. 1 replicate for each concentration without algae.

GROWTH MEDIUM
- Standard medium used: Yes, M1 medium used for stock culture.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA))
- Culture medium different from test medium: yes, propagation in M1 medium, test in M2 medium and a pre-culture was established in M2 medium 4 days before test initiation. M2 test medium was slightly modified by adding additional NaHCO2 and adjusting pH to 7.0 to enable better algal growth in a closed system.

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: continuous illumination using 30 Watt 'Cool-white' TLD-Iamps. Light intensity within the range of 79 to 91 mE/(m²∙s).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beginning of the test, cells were counted with a microscope and counting chamber. Thereafter cell densities were measured using a spectrophotometer with immersion probe (wavelength 720 nm, path length = 10 mm). At the end of the study final microscopic observations were made on the control and highest test concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Yes, test was a combination limit/range-finding test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The ErC50 result corresponds to a 100% water accommodated fraction of MeDONA (loading rate of 100 mg/L).

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes, see Figure 1
- Growth in controls (validity criteria):
- cell density increase through 72-hours: factor of >16 after 48 hours
- % coefficient of variance in average specific growth rate at hour 48: 4% (Table 1)
- % coefficient of variance in section-specific growth rates to hour 48: 8% (Table 2)

Any visual signs of phytotoxicity (abnormalities): No abnormalities were noted in the report.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test substance has low solubility, is hydrolytically unstable and likely to be volatile.
- Effect concentrations exceeding solubility of substance in test medium: yes
-Other: The pH in the controls at the end of the study was 9.1 which exceeded the upper limit of 9.0. Cells were still in active log growth at study conclusion; therefore the study integrity was not adversely affected

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-h ErC50: 1.6 mg/L, 95% C.I. 1.1 - 2.3 mg/L. The historical ranges for growth rate reduction for Pseudokirchneriella subcapitata lie between 0.82 and 2.3 mg/l. Hence, the growth rate reduction EC50: 72h for the Pseudokirchneriella subcapitata tested corresponds with this range.

Reported statistics and error estimates:

No EC50 values could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration). EC50 based on growth rate and biomass >100 mg/L

Any other information on results incl. tables

Table 1 Cell Density, Yield Inhibition and Growth Rate Reductions
Test Group¹ (% WAF)	Vessel Number	Cell Densities (10^4cells/mL)			Yield	Yield Inhibition (%)	Growth Rate (µ)	Growth Rate Reduction (%)
		Exposure Period (hours)
		0	24	48	0 - 48h	0 - 48h	0 – 48h	0 – 48h
	1	1	4.80	27.02	26.02		0.06868
	2	1	6.11	27.74	26.74		0.06923
	3	1	5.88	26.73	25.73		0.06845
	4	1	5.29	24.44	23.44		0.06659
Control	5	1	5.35	21.78	20.78		0.06419
	6	1	4.82	21.36	20.36		0.06378
	Mean				23.85		0.06682
							CV  4%
	1	1	5.04	26.57	25.57	-7	0.06833	-2
0.10	2	1	4.87	25.76	24.76	-4	0.06768	-1
	3	1	5.53	25.96	24.96	-5	0.06784	-2
	Mean				25.10	-5.2	0.06795	-1.7
	1	1	5.10	28.16	27.16	-14	0.06954	-4
1.0	2	1	4.21	24.31	23.31	2	0.06648	1
	3	1	5.12	28.19	27.19	-14	0.06956	-4
	Mean				25.89	-8.6	0.06853	-2.6
	1	1	6.11	28.40	27.40	-15	0.06972	-4
10	2	1	5.13	28.25	27.25	-14	0.06961	-4
	3	1	5.07	25.10	24.10	-1	0.06714	0
	Mean				26.25	-10.1	0.06882	-3.0
	1	1	2.07	26.75	25.75	-8	0.06847	-2
	2	1	2.85	28.29	27.29	-14	0.06964	-4
	3	1	1.93	25.41	24.41	-2	0.06740	-1
100	4	1	2.08	25.74	24.74	-4	0.06767	-1
	5	1	3.14	26.71	25.71	-8	0.06844	-2
	6	1	2.22	25.98	24.98	-5	0.06786	-2
	Mean				25.48	-6.9	0.06825	-2.1
1) Test groups represent percentages of a water accommodated fraction (WAF) prepared at 100 mg/L.

Table 2 Growth rate: section-by-section
Test Group¹ (% WAF)	Vessel Number	Growth rate (µ)		Growth rate reduction (%)
		0-24h	24-48h	0-24h	24-48h
	1	0.06536	0.07200
	2	0.07541	0.06304
	3	0.07381	0.06309
Control	4	0.06941	0.06377
	5	0.06988	0.05850
	6	0.06553	0.06203
	Mean	0.06990	0.06374
	CV	6%	7%
	The mean CV for sec-by-sec specific growth rate was: 8%
	1	0.06739	0.06927	4	-9
0.10	2	0.06596	0.06941	6	-9
	3	0.07126	0.06443	-2	-1
	Mean	0.06820	0.06770	2.4	-6.2
	1	0.06789	0.07119	3	-12
1.0	2	0.05989	0.07306	14	-15
	3	0.06805	0.07108	3	-12
	Mean	0.06528	0.07178	6.6	-12.6
	1	0.07541	0.06402	-8	0
10	2	0.06813	0.07108	3	-12
	3	0.06764	0.06665	3	-5
	Mean	0.07039	0.06725	-0.7	-5.5
	1	0.03031	0.10662	57	-67
	2	0.04364	0.09563	38	-50
	3	0.02740	0.10740	61	-69
100	4	0.03052	0.10482	56	-64
	5	0.04768	0.08920	32	-40
	6	0.03323	0.10249	52	-61
	Mean	0.03546	0.10103	49.3	-58.5
A) Test groups represent percentages of a water accommodated fraction (WAF) prepared at 100 mg/L.

 

Table 3 Concentration of MeDONA in test medium
Time of Sampling (hours)	Concentration
	Percentage of WAF^A(%)	Analyzed^C(mg/L)
	0	< 0.1
	0	< 0.1
	0.1	< 0.1
	0.1	< 0.1
	1.0	< 0.1
0	1.0	< 0.1
	10	< 0.1
	10	< 0.1
	100	< 0.1
	100	< 0.1
	100^B	< 0.1
	100^B	< 0.1
	0	< 0.1
	0	< 0.1
	0.1	< 0.1
	0.1	< 0.1
	1.0	< 0.1
24	1.0	< 0.1
	10	< 0.1
	10	< 0.1
	100	< 0.1
	100	< 0.1
	100^B	< 0.1
	100^B	< 0.1
A) A water accommodated fraction (WAF) prepared at the loading rate of 100 mg/L. B) Without algae C) Response in the test samples was smaller than the response of the recovery samples at target concentration of 0.1 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Cell density increased in 48 hours by average factor >16. Control cultures mean coefficient of variation sec-by-sec specific growth rates < 35%. Control cultures coefficient of variation of specific growth rates during whole test < 7%.

Conclusions:

The 48-hour ErC50 of MeDONA to Pseudokirchneriella subcapitata is > 100 mg/L. The 48-hour NOErC of MeDONA to Pseudokirchneriella subcapitata is 100 mg/L.

Executive summary:

The toxicity of MeDONA to the green algae, Pseudokirchneriella subcapitata, was assessed in a 48-hour toxicity test conducted according to the OECD 201 (2006) method. Exposure period was reduced to 48 hours due to the study being conducted in completely sealed test chambers.Exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met. No reduction in rate or inhibition of yield was recorded up to and including the highest concentration tested of MeDONA after 48 hours. The 48-hour ErC50 of MeDONA to Pseudokirchneriella subcapitata for growth rate was > 100 mg/L, corresponding to the 100% Water Accommodated Fraction (WAF). Concentration levels that might have been toxic for algae could not be reached due to the behavior of MeDONA in water (low solubility, volatile and subject to hydrolysis). This study is considered reliable with restrictions due to the unstable nature of the compound. The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. Therefore, the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis. Results Synopsis

Test Type: static (based on data obtained using OECD 201 methodology)

48-hr ErC50: > 100 mg/L

48-hr NOErC: 100 mg/L

The ErC50 and NOErC results correspond to a loading rate of 100 mg/L MeDONA.