Chlorobenzene

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:

2011-02-08 till 2011-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Water solubility: 0.207 g/L (according to data of the sponsor)
Density: 1.106 kg/L (according to data of the sponsor)
Vapour pressure: 11.7 hPa (20°C) (according to data of the sponsor)
Stability of test concentration/s during exposure: Examined by chemical analysis (HPLC) at 0, 24, 48 and 72 hours.

Analytical monitoring:

yes

Details on sampling:

- Sampling Schedule: Test concentration of 100 mg/L and control concentration of 0 mg/l were both measured at 0, 24, 48 and 72 h.
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.

Vehicle:

no

Details on test solutions:

A stock solution was prepared to give the desired series of test concentrations. To achieve this 90.4 μL (equals 100 mg/L) of the test item were added to 1 litre of dilution water and treated for 1 h in an ultrasonic bath and afterwards stirred for 1.5 h on a magnetic stirrer. The pH was measured to be 7.5.

To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.515 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. To avoid evaporation effects all flasks were sealed with glass stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name : Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source : Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures : Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures : The algal inocula for a test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

1.3 °dH (22.5 mg/L CaCO3)

Test temperature:

21-24 °C +/- 2 °C

pH:

Control: 8.0 at 0 h and 10.5 at 72 h
100 mg/L : 7.6 at 0 h and 7.9 at 72 h

Nominal and measured concentrations:

Test concentrations: 6.3, 12.5, 25, 50 and 100 mg/L plus control
The results are expressed in terms of measured initial concentrations. Effective concentrations ranged from 31.0 % to 55.8 % of nominal values at 0 hours, from 22.2 % to 50.2 % of nominal values at 24 hours, from 20.8 % to 50.8 % of nominal values at 48 hours and from 40.4 % to 50.1 % of nominal values at 72 hours.
Based on initial concentrations the recovery rates after 72 hours ranged from 87.2 % to 90.1 % of initial values, indicating a good stability of the test item in the closed test system.

Details on test conditions:

- Test vessels : 300 mL Erlenmeyer flasks with glass stoppers, test volume: 100 mL
- Culturing apparatus : Light chamber in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily in a water filled flask which was incubated under the same conditions as the test flasks.
- Light intensity : At the average of the test solutions, a light intensity in the range 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was used. Light intensity was checked before the start of the study.
- Cell density measurements : Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.
- Experimental design : 5 test concentrations plus 1 control 1 additional replicate for analytical purposes for each test item concentration and control.
Additionally, highest test concentration without algae.
Initial cell density in the test cultures approximately 5000 cells per millilitre.
- Test item concentration/s : 6.3, 12.5, 25, 50 and 100 mg/L
- Method of administration : stock solution
- Duration of exposure : 72 hours
- Criteria of effects : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 9.8 - 13.3 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 3.7 -7.2 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CL: 7.9 - 8.3 mg/L

Duration:

72 h

Dose descriptor:

other: EyC10

Effect conc.:

5.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 5.7 - 6.1 mg/L

Duration:

72 h

Dose descriptor:

other: NOEC [y]

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: LOEC [y]

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The substance was tested in the following concentrations: 6.3, 12.5, 25, 50 and 100 mg/l
Sample ware taken at the following time points: 0, 24, 48, 72 h

Mean specific growth rate in Desmodesmus subspicatus as Dependent on Concentration and Time

Tab. 1: Mean specific growth rate in Desmodesmus subspicatus as dependent on concentration of the test item and time; Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates; CV: coefficient of variation (calculated from InputRawData)

Treatment (mg/l)	Control	3.329	5.873	11.203	22.316	55.835
0h	0.000	0.000	0.000	0.000	0.000	0.000
	0.000	0.000	0.000	0.000	0.000	0.000
	0.000	0.000	0.000	0.000	0.000	0.000
Mean:	0.000	0.000	0.000	0.000	0.000	0.000
Std. dev:	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
n:	3	3	3	3	3	3
CV:
24h	1.386	1.386	1.386	1.386	0.693	0.693
	1.386	1.386	1.386	0.693	0.693	0.693
	1.386	1.386	1.386	0.693	0.693	0.693
Mean:	1.386	1.386	1.386	0.924	0.693	0.693
Std. dev:	0.0000	0.0000	0.0000	0.4002	0.0000	0.0000
n:	3	3	3	3	3	3
CV:	0.0	0.0	0.0	43.3	0.0	0.0
48h	1.386	1.445	1.242	0.896	0.347	0.347
	1.386	1.386	1.242	0.693	0.347	0.347
	1.445	1.386	1.242	0.693	0.347	0.347
Mean:	1.4406	1.406	1.242	0.761	0.347	0.347
Std. dev:	0.0340	0.0340	0.0000	0.1170	0.0000	0.0000
n:	3	3	3	3	3	3
CV:	2.4	2.4	0.0	15.4	0.0	0.0
72h	1.365	1.342	1.330	0.597	0.231	0.231
	1.342	1.365	1.317	0.597	0.231	0.231
	1.353	1.365	1.317	0.597	0.231	0.231
Mean:	1.353	1.357	1.321	0.597	0.231	0.231
Std. dev:	0.0115	0.0133	0.0073	0.0000	0.0000	0.0000
n:	3	3	3	3	3	3
CV:	0.8	1.0	0.5	0.0	0.0	0.0

Effective Concentrations (ECx) for Growth Rate at 72 h

Growth Rate of Desmodesmus subspicatus: Tab. 2: %Inhibition caused by the test item after 72 h.

 

Treatment (mg/l)	Mean	Std Dev	n	% Inibition
Control	1.353	0.0115	3	0.0
3.329	1.357	0.0133	3	-0.3
5.873	1.321	0.0073	3	2.4
11.203	0.597	0.0000	3	55.9
22.316	0.231	0.0000	3	82.9
55.835	0.231	0.0000	3	82.9

table 3 Analysis:

Test item concentration (mg/l)	HPLC values (mg/L)		HPLC values (mg/L)
	0h	24h	48h	72h
Control	<0.033	<0.033	<0.033	<0.033
6.3	3.329	2.992	2.754	2.904
12.5	5.873	5.260	5.0620	5.272
25	11.203	10.223	9.829	10.095
50	22.316	19.774	17.437	20.224
100	55.835	50.180	49.055	50.050
100 without algae	30.964*	22.206*	20.831*/50.753	46.534

* The test assay without algae was produced with only 50 mL test volume instead of 100 mL. The solution was prepared in 300 mL Erlenmeyer flasks. Obviously the enhanced headspace led to a higher evaporation effect of the volatile test item. For this reason analysis after 48 h and 72 h of exposure was made using the replicate of the test (test volume 100 mL) instead of the additional replicate for analytical purposes. The results after 48 h and 72 h showed comparable values to those of the test item concentration 100 mg/L with algae. This indicates a correct concentration of the test item during the complete test.

 

table 4. pH values at the start and the end of the study

Test item concentration (mg/L)	pH values
	0h	72h
Control	8.0	10.5
6.3	8.0	10.5
12.5	7.9	10.5
25	7.9	8.6
50	7.6	8.1
100	7.6	7.9

 

Validity criteria fulfilled:

yes

Remarks:

(-The factor of biomass parameter 58.0 >16; - The mean of the replicate coefficients of variation in the section-by-section growth: 7.0 %. <35 %; - The coefficient of variation of the mean specific growth rate replicates in the control 0.8 % < 7%.)

Conclusions:

The toxicity to algae of chlorobenzene was determined with ErC50 and ErC10 values at 11.4 and 5.8 mg/L, respectively. A NOEC of 3.3 mg/L and a LOEC of 5.9 mg/L were also calculated, based on growth rate.

Executive summary:

In order to test toxicity to algae of chlorobenzene, Desmodesmus subspicatus was exposed to the test solution of five nominal concentrations of the test substance (6.3, 12.5, 25, 50 and 100 mg/L) and a blank control solution for a period of 72 h under static conditions, and according to OECD Guideline 201 (Alga, Growth Inhibition Test). The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. The measured concentrations confirmed that deviation from the nominal concentrations was less than 30 % (measured concentrations were in the range of 87.2 - 90.1 % of nominal concentrations). The toxicity to algae of chlorobenzene was determined with ErC50 and ErC10 values at 11.4 and 5.8 mg/L, respectively. A NOEC of 3.3 mg/L and a LOEC of 5.9 mg/L was also calculated, based on growth rate. 

Description of key information

72h-ErC50 (Desmodesmus subspicatus) = 11.4 mg/L; According to OECD TG 201; Neuhahn (2011).
72h-ErC10(Desmodesmus subspicatus) = 5.8 mg/L; According to OECD TG 201; Neuhahn (2011).

Key value for chemical safety assessment

EC50 for freshwater algae:

11.4 mg/L

EC10 or NOEC for freshwater algae:

5.8 mg/L