1-ethoxy-2,3-difluorobenzene

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:

2000-03-23 till 2000-03-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted June 07, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

December 29, 1992

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

For the analytical measurement a sample from the freshly prepared test medium was taken at the start of the study. For the determination of the maintenance of the test material concentration during the exposure period a sample was taken from the medium at the end of the exposure period. Analyses were performed with test material concentrations of 2, 10 and 47 mg/L.

Vehicle:

no

Details on test solutions:

The test media (reconstituted water and test material) were freshly prepared. A stock preparation with a test material concentration of 0.1 g/L was prepared. Therefore, the calibrated flask with the test material and the vehicle, reconstituted water, was treated with an Ultra Thurrax and then in an ultrasonic device for 60 minutes. The stock preparation was diluted with reconstituted water to the different test medium concentrations.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: No. SAG 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universitat Göttingen

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

252 mg CaCO3/L

Test temperature:

23 to 24°C

pH:

7.59 - 7.76

Nominal and measured concentrations:

Nominal concentrations: 2, 4, 10, 21 and 47 mg/L
Measured concentrations: Please see Table 1 below.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed (ground stopper)
- Material, fill volume: glass, 100 mL
- Aeration: no
- Initial cells density: 10,000 algae cells/mL
- Control end cells density: 606,844 algae cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: According to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: 7300 to 9000 Lux (fluorescent tubes: Osram dulux 24W/21 - 840)

EFFECT PARAMETERS MEASURED
Algae cell densities were determined by particle counting with an electronic particle counter (Schaerfe System, CASY 1)

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no

Validation criteria:
In the controls, cell density increased by an average factor of > 16 within 3 days. Further, all test conditions remained within the ranges prescribed by the protocol.

In the two highest test material concentrations, the analytically determined test material concentration at the start and the end of the experimental part was about 52 % of the nominal value. The analytical test material concentration at nominally 2 mg/L was below the limit of quantification. At the end of the study, the analytical concentrations were the same at the beginning of the experimental part. Thus the test material concentration could maintained as at > 80 % of the initial concentration throughout the experimental part.

Reported statistics and error estimates:

The NOEC was determined as the highest dose without inhibitory effect. The EC50 calculation was carried out with the PC-program 511.

Table 1: Measured concentrations

Nominal concentration [mg/L]	Analytical concentration [mg/L]
	After 0h		After 72h
2	< 2			< 2
10	5	53.2%		5	53.2 %
47	25	50.0 %		25	50.0 %

 

 

Table 2: Cell density and growth inhibition based on nominal concentrations

 

Nominal concentration [mg/L]	Cell density per mL		mean value	Growth inhibition (0-72 hours)
	0 hour	72 hours	72 hours
Control	10,000	587,265	606,844
	10,000	609,882
	10,000	623,385
2	10,000	503,685	508,732	16.8
	10,000	509,040
	10,000	513,471
4	10,000	526,743	534,534	12.4
	10,000	550,956
	10,000	525,903
10	10,000	519,750	501,816	17.9
	10,000	496,755
	10,000	488,943
21	10,000	156,328	156,053	75.6
	10,000	146,544
	10,000	165,288
47	10,000	15,584	13,792	99.4
	10,000	13.288
	10,000	12,504

 

Validity criteria fulfilled:

yes

Conclusions:

An aqueous solution of the test item in an static system revealed an aquatic toxicity towards algae: Growth rate after 72h: EC50: 6.24 mg/L, NOEC: 1.04 mg/L. Biomass after 72h: EC50: 5.1 mg/L, NOEC: 1.04 mg/L.

Executive summary:

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested according to OECD 201 and EU Method C.3. Due to the high volatilization and recovery of the test material it was necessary to include some changes of the regular guidelines in this study. Therefore 100 mL Erlenmeyer flasks were filled with 100 mL test medium and sealed to form a closed test system to avoid loss of test material during the exposure period due to volatilization. The algae were exposed to different concentrations of test material preparations and the growth was calculated after 72 hours exposure in the test medium. Based on the results of a pretest, the main study with the test item was performed with nominal concentrations of 2, 4, 10, 21, and 47 mg test material/L reconstituted water. The study design included three replicates per test material concentration, each containing about 100 mL test medium and 10,000 cells/mL at the start of the experimental phase and a control without algae. The control analysis showed a recovery of about 52 % for the two highest nominal test concentrations of 10 and 47 mg/L at the start and the end of the study. At the nominal concentration of 2 mg/L, the analytically determined test material concentration was below the limit of quantification. Therefore, the test results are related to the nominal concentration of the test material. At the end of the study, the analytical concentrations were the same as at the beginning of the experimental part. Thus the test material concentration could be maintained at > 80 % of the initial concentration throughout the experimental part. The median effective concentration EC₅₀ which would result in a 50 % growth inhibition compared to the control was calculated for the measured concentration of the test item to be 6.24 mg/L, the NOEC is 1.04 mg/L. For biomass, the EC₅₀ was calculated to be 5.1 mg/L, the NOEC 1.04 mg/L.

Description of key information

An aqueous solution of the test item in an static system revealed an aquatic toxicity towards algae: Growth rate after 72h: EC50: 6.24 mg/L, NOEC: 1.04 mg/L. Biomass after 72h: EC50: 5.1 mg/L, NOEC: 1.04 mg/L (reference 6.1.5 -1).

Key value for chemical safety assessment

EC50 for freshwater algae:

6.24 mg/L

EC10 or NOEC for freshwater algae:

1.04 mg/L

Additional information

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested according to OECD 201 and EU Method C.3. Due to the high volatilization and recovery of the test material it was necessary to include some changes of the regular guidelines in this study. Therefore 100 mL Erlenmeyer flasks were filled with 100 mL test medium and sealed to form a closed test system to avoid loss of test material during the exposure period due to volatilization. The algae were exposed to different concentrations of test material preparations and the growth was calculated after 72 hours exposure in the test medium. Based on the results of a pretest, the main study with the test item was performed with nominal concentrations of 2, 4, 10, 21, and 47 mg test material/L reconstituted water. The study design included three replicates per test material concentration, each containing about 100 mL test medium and 10,000 cells/mL at the start of the experimental phase and a control without algae. The control analysis showed a recovery of about 52 % for the two highest nominal test concentrations of 10 and 47 mg/L at the start and the end of the study. At the nominal concentration of 2 mg/L, the analytically determined test material concentration was below the limit of quantification. Therefore, the test results are related to the nominal concentration of the test material. At the end of the study, the analytical concentrations were the same as at the beginning of the experimental part. Thus the test material concentration could be maintained at > 80 % of the initial concentration throughout the experimental part. The median effective concentration EC₅₀ which would result in a 50 % growth inhibition compared to the control was calculated for the measured concentration of the test item to be 6.24 mg/L, the NOEC is 1.04 mg/L. For biomass, the EC₅₀ was calculated to be 5.1 mg/L, the NOEC 1.04 mg/L (reference 6.1.5-1).