2,2-Difluoroethyl Acetate

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:

From 04 January 2016 to 08 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L nominal (limit test).
- Sampling method: 10-mL samples were collected in all test solutions from the middle layer of the vessels at the beginning of exposure and at 24, 48, and 72 hours after the beginning of exposure.
- Sample storage conditions before analysis: No data available.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test item (100 mg) was dissolved in 1000 mL test water to give a 100 mg/L stock solution from which the test solution was made. The stock solution was stirred for 5 minutes with a magnetic stirrer to ensure adequate mixing and homogeneity.
- Controls: Only dilution water.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater unicellular green algae.
- Strain: ATCC22662.
- Source: American Type Culture Collection (Receipt: June 20, 1996).
- Method of cultivation: Algae have been maintained by period subculture using Gorham medium. Algae were kept under sterile condition. Sterility test has been performed periodically, every six months, to confirm that the algae were not contaminated.

ACCLIMATION
- Preculture period: From January 1 to 5, 2016.
- Culturing media and conditions: Temperature: 22±2°C; Light intensity: 65 to 75 μE/m2/s; Revolution: 100 rpm.
- Any deformed or abnormal cells observed: Neither modified nor unusual cells were observed.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

No data available.

Test temperature:

21.6°C to 22.2 °C.

pH:

Control: 7.8 to 10.3.
100 mg/L: 7.8 to 9.7.
N.B. The pH of the control group increased more than 1.5 units during the test. The concentration of bicarbonate ion was decreased by carbon dioxide assimilation in algae and there was very little air exchange (supply of carbon dioxide) in closed system, and therefore, the pH was increased.

Dissolved oxygen:

No data available.

Salinity:

Not concerned.

Conductivity:

No data available.

Nominal and measured concentrations:

-Nominal concentations: control and 100 mg/L.
-Mesured concentration on Day 0 (start of experiment): <0.1 and 98.1 mg/L.
-Mesured concentration after 24h: <0.1 and 91.2 mg/L.
-Mesured concentration after 48h: <0.1 and 87.2 mg/L.
-Mesured concentration after 72h: <0.1 and 36.1 mg/L.
-Time-weighted mean measured concentration: <0.1 and 80.6 mg/L.
N.B. Hydrolysis of the test substance and/or metabolism of the test substance by algae, etc., were considered as a reason for the concentration decrease over 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask with stopper.
- Type (delete if not applicable): closed.
- Fill volume: 100 mL/vessel.
- Aeration: no but shaking (100 rpm).
- Initial cells density: 5x10E3 cells/mL.
- Control end cells density: 517x10E3 cells/mL.
- No. of vessels per concentration (replicates): 6.
- No. of vessels per control (replicates): 6.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium.
- Intervals of water quality measurement: pH was measured at the beginning and at the end of exposure. During the exposure period, temperatures, light intensities, and revolutions in the culturing apparatus were measured at least once a day.

OTHER TEST CONDITIONS
- Sterile test conditions: aseptic technique.
- Adjustment of pH: no.
- Photoperiod: continuous illumination with white fluorescent lamp (on the surface of test culture).
- Light intensity and quality: 60 to 65 μE/m2/s.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the exposure, biomass in the inoculum culture was determined with an electronic particle counter (CDA-500, Sysmex Co., Ltd. ) as well as microscopically using a hemacytometer (ECLIPSE TE300, Nikon Co., Ltd.). Then, the biomass of algae in each test vessel was measured at 24, 48 and 72 hours after the beginning of exposure. One milliliter of test culture was suspended in 9.0 mL of electrolyte to count the algal cells by electronic particle counter (CDA-500, Sysmex Co., Ltd. ).
- Chlorophyll measurement: no.
- Growth rate: Growth rate was calculated from biomass determinations and then used to determined percent inhibition of growth rate.

TEST CONCENTRATIONS
- Limit test at 100 mg/L.
- Range finding study:
* Test concentrations: control, 0.1, 1, 10 and 100 mg/L nominal.
* Results used to determine the conditions for the definitive study: No growth inhibition, therefore a limit design was applied for the definitive test.

CULTURING APPARATUS
-Details on culturing apparatus used: AGP-150RL, Itoh Seisakusho, Ltd.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 80.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 80.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes, the biomass in the control cultures increased exponentially of 103 fold during 72-hour culture (cell density: 5x10E3 at the beginning of the test and 517x10E3 at the end of the test).
- Observation of abnormalities: By microscopic observation at the end of exposure, neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed in the treatment exposed to 100 mg/L, and the algae looked normal compared with that in the control group.
- Unusual cell shape: no.
- Colour differences: In the control and the treatment exposed to 100 mg/L, the colour of the test cultures (as observed with the naked eye) showed a tendency to get more greenish during the exposure period due to the algal population growth.
- Flocculation: no.
- Adherence to test vessels: no.
- Aggregation of algal cells: no.
- Effect concentrations exceeding solubility of substance in test medium: no.

Results with reference substance (positive control):

- Results with reference substance is valid? Yes (within the average values of ErC50 found in the testing laboratory sinceJune, 2000: average ErC50 ± standard deviation = 0.814 ± 0.0723mg/L, n=32, minimum-maximum = 0.687-0.965 mg/L).
- 72-hour ErC50: 0.809 mg/L (95% confidence limits: 0.789-0.831 mg/L, Exposure period: From December 25 to 28, 2015).

Reported statistics and error estimates:

The ErC50 value and associated 95% confidence limits could not be determined by least squares linear regression analysis because the test was conducted at one concentration level showing no significant growth inhibition. Therefore, the ErC50 was set superior to the highest concentration tested (i.e. 100 mg/L nominal, 80.6 mg/L actually measured).
The NOECr (0-72h) value was determined by Student’s t-test, subsequent to F test for homogeneity of variances. Statistical analyses were performed using Yukms Statlight #3 software (Yukms Corp., Tokyo) and all tests of significance were at α=0.05.

Biomass of Pseudokirchneriella subcapitata during the 72-Hour Exposure:

Test Group	Nominal concentration [Mean^ameasured concentration] (mg/L)	Vessel No.	Biomass (cells/mL)
			0 Hour*	24 Hours	48 Hours	72 Hours
Control	--	1	5000	29600	156000	509000
		2	5000	31600	152000	496000
		3	5000	28600	165000	532000
		4	5000	27700	175000	519000
		5	5000	31600	179000	529000
		6	5000	28000	157000	514000
		Average	5000	29500	164000	517000
		SD	0	1740	11000	13300
Conc.1	100 [ 80.6 ]	1	5000	28800	172000	873000
		2	5000	30400	198000	933000
		3	5000	31600	192000	906000
		4	5000	31700	170000	918000
		5	5000	30100	168000	835000
		6	5000	31400	188000	878000
		Average	5000	30700	181000	891000
		SD	0	1130	12900	35600

a: Time weighted mean;  SD: Standard deviation; *  : Nominal initial biomass.

 

 

Growth Inhibitions (%) of Pseudokirchneriella subcapitata:

Test Group	Nominal Concentration [Mean^a measured concentration] (mg/L)	Vessel No.	Growth rate
			Rate μ(0-72h)	Inhibition(%)^*1 Iμ(0-72h)
Control	--	1	0.0642
		2	0.0638
		3	0.0648
		4	0.0645
		5	0.0647
		6	0.0643
		Average	0.0644	-
		SD	0.0004
Conc.1	100 [ 80.6 ]	1	0.0717	-11.3
		2	0.0726	-12.7
		3	0.0722	-12.1
		4	0.0724	-12.4
		5	0.0711	-10.4
		6	0.0718	-11.5
		Average	0.0720^++	-11.7
		SD	0.0005

a : Time weighted mean

*1: Values are the growth inhibition (%) relative to the control. Negative values correspond to growth stimulation as compared to control.

SD: Standard deviation.

++:  Although growth rates were significantly different between the control group and the group exposed to 100 mg/L (++:α=0.01), this is representative of a growth stimulation. 

  

 

Growth rates of control:

Vessel No.	Growth rates					SD	CV (%)
	μ(0-72h)	μ(0-24h)	μ(24-48h)	μ(48-72h)	Average of μ (0-24h, 24-48h, 48-72h)
1	0.0642	0.0741	0.0693	0.0493	0.0642	0.0132	20.6
2	0.0638	0.0768	0.0654	0.0493	0.0638	0.0138	21.6
3	0.0648	0.0727	0.0730	0.0488	0.0648	0.0139	21.5
4	0.0645	0.0713	0.0768	0.0453	0.0645	0.0168	26.0
5	0.0647	0.0768	0.0723	0.0452	0.0648	0.0171	26.4
6	0.0643	0.0718	0.0718	0.0494	0.0643	0.0129	20.1
Average	0.0644	0.0739	0.0714	0.0494	-	-	22.7
SD	0.0004	0.0024	0.0038	0.0021	-	-	-
CV (%)	0.6	3.2	5.3	4.4	-	-	-

SD: Standard deviation.

CV: Coefficient of variation.

 

 

Validity criteria:

-The factor of biomass increase in the controls is greater than 16. Indeed the average biomass is 5x10E3 cell/mL at 0 hour and 517x10E3 cell/mL at the end of the test.

-The mean coefficient of variation for section-by-section specific growth rates in the controls is =< 35% with a calculated value of 22.7%. 

-The coefficient of variation of average specific growth rates during the whole test period in replicate controls is less than 7%. Indeed the highest coefficient of variation during the whole test period in replicate control cultures is 5.3%.

All validation criteria for the study were satisfied.

 

Validity criteria fulfilled:

yes

Remarks:

All validation criteria for the study were satisfied (see above).

Conclusions:

The 72-hour ErC50 and 72-hour NOErC of 2,2-Difluoroethyl acetate to Pseudokirchneriella subcapitata were determined to be > 80.6 mg/L and ≥ 80.6 mg/L, respectively.

Executive summary:

The toxicity of 2,2-Difluoroethyl acetate to Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD guideline 201 under GLP compliance.

Algal cultures were exposed for 72 hours under static conditions to a control treatment and to a nominal limit test concentration of 100 mg/L 2,2-Difluoroethyl acetate (six replicates in each case). The corresponding actual concentration was measured using gas chromatography/mass spectrometry (GC/MS) as being equal to 80.6 mg/L (time weighted average of concentrations measured in samples taken at the beginning of the test and after 24, 48 and 72 hours). Algal cell densities were measured after 24, 48 and 72 hours using an electronic particle counter and inhibition of growth rate was calculated.

In the treatment exposed to the limit test concentration, the algal growth rate was significantly different from the one in the control treatment. However, this significant effect reflected a growth stimulation (+ 11.7 %) rather than an inhibition.

Therefore, the 72-hour EC50 and 72-hour NOEC based on growth rate inhibition in P. subcapitata were concluded to be > and ≥ to the limit test concentration of 80.6 mg/L, respectively.

Description of key information

The 72-hour ErC50 and 72-hour NOErC of 2,2-Difluoroethyl acetate to Pseudokirchneriella subcapitata based on growth rate were determined to be > 80.6 mg/L and ≥ 80.6 mg/L, respectively. 

Additional information

An experimental study performed under GLP compliance and in accordance with OECD test guideline 201 was flagged as a key study and assigned a Klimisch score of 1. It reports a stimulation effect of 2,2-Difluoroethyl acetate on the growth of Pseudokirchneriella subcapitata at the limit test concentration of 80.6 mg/L. Therefore, The 72-hour ErC50 and 72-hour NOErC of 2,2-Difluoroethyl acetate to Pseudokirchneriella subcapitata based on growth rate were determined to be > 80.6 mg/L and ≥ 80.6 mg/L, respectively.