1-bromo-3,4,5-trifluorobenzene

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:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

1989

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 5.6, 10, 18, 32, 56, 100 mg/L
- Sampling freency: First test: at t=0 h and t=24 h; Second test: at 0, 24, 48 and 72 hours.
- Sample storage conditions before analysis: Not applicable, samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation during the range-finding test and the EC50 tests started with a stock solution at nominal 100 mg/L. The test substance did not easily dissolve at this concentration. Magnetic stirring had to be applied for 10 to 15 minutes. Since this did not result in complete dissolving of the test substance, in addition, ultra sonic treatment had to be applied for 10 to 15 minutes. The various test concentrations were prepared by subsequent dilution of the stock solution of 100 mg/L and stirred to ensure complete mixing. All test solutions were prepared and tested in completely sealed glass ware to minimize volatisation of the test substance. All final test solutions were clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, specific volumes of an algal suspension were added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

pH:

7.7 - 9.8

Nominal and measured concentrations:

Nominal: up to 100 mg/L
Measured concentration: As in 100 mg/L solution the concentration loss was below 20%, the final test was performed without analytical control. The range of exposure conecentrations in the final test are estimated to correspond with 2.0, 3.6, 6.5, 12, 20 and 49 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL
- Type: closed (sealed with a glass cap)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Initial cells density: 10^4 cells/mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium according to ISO 8692
- Intervals of water quality measurement: pH measured at beginning and end of test, temperature measured daily

- Photoperiod: continuous
- Light intensity and quality: 7500 - 8000 lux

EFFECT PARAMETERS MEASURED
At the beginning of the test cells were counted by microscope, using counting chamber. Thereafter, cell densities were determined by spectrophotometry measurement of samples at 720 nm using a Lambdan Spectrophotometer (Perkin-Elmer, Illinois, USA), with a cuvette of 5 path-length. Algal medium was used as blank.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1 - 100 mg/L, factor 10
- Results used to determine the conditions for the definitive study: EC50 10 - 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.2 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 4.6 - 18 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.4 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 1.2 - 4.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 9.7 - 34 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.8 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 2.0 - 7.1 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

%Concentration loss over test: 19 ... 33

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Results:
The 72-hour EC50 for growth inhibition was estimated at 1.1 mg/L.
The 72-hour EC50 for growth rate reduction was 1.4 mg/L with a 95% confidence interval of 0.44 - 4.3 mg/L.

Acceptability of the test:

1. In the controls, cell density increased by an average factor of > 16 within 3 days.

2. Analysis of samples taken from the the solution without algae showed that the actual exposure concentration could be maintained fairly stable during the test period in spite of the volatility of the test substance. The exposure was based on the average measured concentrations.

3. Further, all test conditions remained within the ranges prescribed by the protocol as far as exposure in airtight vessels allowed. The increase of pH by more than 1.5 unit could not be prevented.

Analytical results

Table 1: Concentrations of Art. 270075 in test medium (first EC50 test) 

Time of sampling [days]	Concentration
	Nominal intended [mg/L]	Expected [mg/L]	Actually analysed^1 [mg/L]	Recovery
0	100^2		44.5	45
0	0	0	n.d.	n.a.
	10	4.5	4.52	100
	18	8.0	7.83	98
	32	14	13.5	96
	56	25	24.8	99
	100	45	37.6	84
24	0	0	n.d.	n.a.
	10	4.5	3.46	77
	18	8.0	6.50	81
	32	14	8.92	64
	56	25	19.6	78
	100	45	27.8	62
	100²	45	35.3	78

¹Mean of duplicate analysis. The maximum deviation between the responses was <10% for each sample.

²Without algae

n.d. Not detected

n.a. Not applicable

Table 2: Concentrations of Art. 270075 in test medium (second EC50 test)

Time of sampling [days]	Concentration			Recovery [%]
	Nominal intended [mg/L]	Expected [mg/L]	Actually analysed^1 [mg/L]
0	100^2		54.0	54
0	0	0	n.d	n.a
	5.6	3.0	2.58	86
	10	5.4	4.91	91
	18	9.7	7.97	82
	32	17	14.7	86
	56	30	26.7	89
	100	54	41.6	77
24	0	0	n.d.	n.a.
	5.6	3.0	1.82	61
	10	5.4	4.05	75
	18	9.7	6.84	71
	32	17	11.4	67
	56	30	23.1	77
	100	54	33.7	62
	100^2	54	34.6	64
48	0	0	n.d.	n.a.
	5.6	3.0	1.96	65
	10	5.4	3.82	71
	18	9.7	6.71	69
	32	17	11.5	68
	56	30	23.7^3	79
	100	54	40.1	74
	100^2	54	41.9	78
72	0	0	n.d.	n.a.
	5.6	3.0	1.00	33
	10	5.4	2.15	40
	18	9.7	3.78	39
	32	17	5.58	33
	56	30	11.5	38
	100	54	22.3	41
	100^2	54	38.3	71

¹Mean of duplicate analysis. The maximum deviation between the responses was <10% for each sample.

²Without algae

³Calculated by extrapolation.

n.d. not detected

n.a. not applicable

Exposure concentrations in the main test

The final test was performed without analytical support. The procedure of preparation of the test concentrations was considered to be repeatable, because the difference between the concentrations measured in the solution prepared at 100 mg/L was less than 20%. Further, the toxicological results of the second EC50 test were in agreement with those expected on the basis of the range-finding test. Hence, the initial concentrations were expected to be in the range of those determined during the first and the second EC50 test. The concentration in the solution prepared at 100 mg/L is estimated to be 49 mg/L, i.e. the mean value of 44.5 mg/L in the first EC50 test and 54 mg/L in the second EC50 test. Further, the average exposure concentrations are expected to be 74 % of the initial concentrations, which corresponds with the mean recovery determined in the second EC50 test. Hence, the range of exposure concentrations in the final test are estimated to correspond with 2.0, 3.6, 6.5, 12, 20 and 49 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

A growth inhibition test on Pseudokirchneriella subcapitata was conducted according to OECD 201. The test substance was measured in a 100 mg/L solution, the substance loss was below 20%. The toxicity parameters were based on the exposure concentrations estimated using the analytical results of the previous tests. Under the conditions of this study, the EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and cell growth (EbC50) after 72 hours was 18 mg/L (95% CI 9.7 - 34 mg/L) and 9.2 mg/L (95% CI 4.6 - 18 mg/L), respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and cell growth (EbC10) after 72 hours was 3.8 mg/L (95% CI 2.0 - 7.1 mg/L) and 2.4 mg/L (95% CI 1.2 - 4.9 mg/L), respectively. The NOEC-values for both inhibition of growth rate and cell growth after 72 hours were 2 mg/L.

Executive summary:

The toxicity of the test substance to the freshwater algae Pseudokirchneriella subcapitata was determined in a Growth Inhibition Test according to OECD guideline 201. Pseudokirchneriella subcapitata were exposed to test item concentrations (estimated based on analytical results of previous tests) of 2.0, 3.6, 6.5, 12, 20 and 49 mg/L and a control. The initial cell density was 10^4 cells/mL. The total test period was 72 hours. The final test was performed without analytical support.
Analysis of samples taken from the solution without algae during previous tests showed that the actual exposure concentration could be maintained fairly stable during the test period in spite of the volatility of the test substance. The toxicity parameters were based on the exposure concentrations estimated using the analytical results of the previous tests.
Under the conditions of this study, the EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and cell growth (EbC50) after 72 hours was 18 mg/L (95% CI 9.7 - 34 mg/L) and 9.2 mg/L (95% CI 4.6 - 18 mg/L), respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and cell growth (EbC10) after 72 hours was 3.8 mg/L (95% CI 2.0 - 7.1 mg/L) and 2.4 mg/L (95% CI 1.2 - 4.9 mg/L), respectively. The NOEC-values for both inhibition of growth rate and cell growth after 72 hours were 2 mg/L.

Description of key information

A growth inhibition test on Pseudokirchneriella subcapitata was conducted according to OECD 201. The test substance was measured in a 100 mg/L solution, the substance loss was below 20%. The toxicity parameters were based on the exposure concentrations estimated using the analytical results of the previous tests. Under the conditions of this study, the EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and cell growth (EbC50) after 72 hours was 18 mg/L (95% CI 9.7 - 34 mg/L) and 9.2 mg/L (95% CI 4.6 - 18 mg/L), respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and cell growth (EbC10) after 72 hours was 3.8 mg/L (95% CI 2.0 - 7.1 mg/L) and 2.4 mg/L (95% CI 1.2 - 4.9 mg/L), respectively. The NOEC-values for both inhibition of growth rate and cell growth after 72 hours were 2 mg/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

18 mg/L

EC10 or NOEC for freshwater algae:

3.8 mg/L

Additional information

The toxicity of the test substance to the freshwater algae Pseudokirchneriella subcapitata was determined in a Growth Inhibition Test according to OECD guideline 201. Pseudokirchneriella subcapitata were exposed to test item concentrations (estimated based on analytical results of previous tests) of 2.0, 3.6, 6.5, 12, 20 and 49 mg/L and a control. The initial cell density was 10^4 cells/mL. The total test period was 72 hours. The final test was performed without analytical support.

Analysis of samples taken from the solution without algae during previous tests showed that the actual exposure concentration could be maintained fairly stable during the test period in spite of the volatility of the test substance. The toxicity parameters were based on the exposure concentrations estimated using the analytical results of the previous tests.

Under the conditions of this study, the EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and cell growth (EbC50) after 72 hours was 18 mg/L (95% CI 9.7 - 34 mg/L) and 9.2 mg/L (95% CI 4.6 - 18 mg/L), respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and cell growth (EbC10) after 72 hours was 3.8 mg/L (95% CI 2.0 - 7.1 mg/L) and 2.4 mg/L (95% CI 1.2 - 4.9 mg/L), respectively. The NOEC-values for both inhibition of growth rate and cell growth after 72 hours were 2 mg/L (reference 6.1.5-1).