Bromobenzene

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:

02 Feb - 05 Feb 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Circular on Test Methods of New Chemical Substances (Japan), Alga, growth inhibition test

GLP compliance:

yes

Remarks:

according to the Japanese GLP Standard

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the refrigerator
- Stability under test conditions: verified by IR
- Solubility and stability of the test substance in the solvent/vehicle: insoluble in water but soluble in alcohol, ether, chloroform, and benzene

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 18, 32, 56 and 100 mg/L
- Sampling method: sample was taken at the beginning of the test, after 24, 48 and 72 hours
- Sample storage conditions before analysis: samples were not stored

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was mixed with medium. After sonication, the stock solution was mixed on a stirrer and stored in a closed bottle. The stock solution was diluted with medium and mixed by hand.
- Controls: medium

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC22662
- Source (laboratory, culture collection): originally obtained from American Type Culture Collection
- Age of inoculum (at test initiation): 5 days
- Method of cultivation: algae were cultured in the Erlenmeyer flasks and shaked

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.5 - 20.7 °C

pH:

7.8 - 10.5

Nominal and measured concentrations:

See table in "Any other information on results incl. tables" below

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass flask, 500 mL
- Aeration: no
- Initial cells density: 5 x 1000 cells/mL
- Control end cells density: 624 x 1000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: not performed
- Photoperiod: continuously
- Light intensity and quality: 60 - 65 μE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
Range finding study:
- Test concentrations: 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: After 72 hours, test substance was between 51 and 62% of the nominal concentration with or without algae

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL

Remarks:

12.0 - 12.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.87 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

ANOVA, Williams test, subsequent to Barlett test

Table 1. Measured concentration of the test substance in test cultures

Nominal concentration (mg/L)	Measured concentration (mg/L) (Percent of nominal)				Mean^ameasured concentration (mg/L) (Percent of nominal)
	0 h	24 h	48 h	72 h
-	< 0.05	< 0.05	< 0.05	< 0.05	-
10	7.55 (76)	4.50 (45)	4.53 (45)	3.89 (39)	4.87 (49)
18	13.8 (77)	8.20 (46)	8.08 (45)	7.30 (41)	8.86 (49)
32	24.8 (78)	14.6 (46)	14.6 (46)	12.4 (39)	15.8 (49)
56	42.9 (77)	25.6 (46)	24.7 (44)	21.1 (38)	27.2 (49)
100	73.9 (74)	43.2 (43)	43.2 (43)	37.6 (38)	46.9 (47)

a: Time weighted mean

Table 2. Biomass of Pseudokirchnerialla subcapitata during the 72 -hour exposure

Nominal concentration [Mean^ameasured concentration] (mg/L)	Biomass (cells/mL) Average ± SD
	0 h*	24 h	48 h	72 h
-	5000 ± 0	2260 ± 1580	18100 ± 25500	624000 ± 48800
10 [4.87]	5000 ± 0	18600 ± 1970	152000 ± 6350	643000 ± 41600
18 [8.86]	5000 ± 0	18200 ± 2020	103000 ± 15900	392000 ± 55400
32 [15.8]	5000 ± 0	12400 ± 500	14400 ± 854	12400 ± 529
56 [27.2]	5000 ± 0	6750 ± 292	6790 ± 470	5930 ± 341
100 [46.9]	5000 ± 0	4960 ± 317	5170 ± 117	4340 ± 635

a: Time weighted mean

SD: Standard deviation

*: Nominal initial biomass

Table 3. Growth inhibition (%) of Pseudokirchneriella subcapitata

Nominal concentration [Mean^ameasured concentration] (mg/L)		Growth rate
		Rate μ(0-72h)	Inhibition (%)^*1 Iμ (0-72h)
-	Average SD	0.0670 0.0011	-
10 [4.87]	Average SD	0.0674 0.0009	-0.6
18 [8.86]	Average SD	0.0605** 0.0019	9.6
32 [15.8]	Average SD	0.126** 0.0006	81.1
56 [27.2]	Average SD	0.0024** 0.0008	96.5
100 [46.9]	Average SD	-0.0021** 0.0020	103.1

a: Time weighted mean

*1: Values are the growth inhibition (%) relative to the control

SD: Standard deviation

**: Indicates a significant difference (α=0.01) from the control

Description of key information

ErC50 (72 h): 12.3 mg/L (measured (TWA), Circular on Methods of New Chemical Substances, Japan).

NOEC (72 h, growth): 4.87 mg/L (measured (TWA), Circular on Methods of New Chemical Substances, Japan).

Key value for chemical safety assessment

EC50 for freshwater algae:

12.3 mg/L

EC10 or NOEC for freshwater algae:

4.87 mg/L

Additional information

There is one GLP study available investigating the toxicity of the test substance to freshwater green algae according to “Circular on Test Methods of New Chemical Substances (Japan)”.

In a static test Pseudokirchneriellea subcapitata were exposed to nominal test concentrations of 10, 18, 32, 56, and 100 mg/L in closed glass flasks for 72 h under controlled conditions. Cell concentrations were measured by electronic particle counter and test solution concentrations were chemically analysed by HPLC, at the beginning of the test and after 24, 48, and 72 h.

Chemical analysis indicated that the test substance was not stable over time. Effect concentrations were provided as time weighted mean values. The derived ErC50 (72 h) was 12.3 mg/L and the NOEC (72 h, growth rate) was 4.87 mg/L.