2-chlorobuta-1,3-diene

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

72 hours

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP-compliant study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

To avoid evaporation of the test item, the Erlenmeyer flasks were closed tightly with glass stoppers. Therefore the pH value decreased more than 1.5 pH units. This minor deviation is not considered to have any impact on the putcome of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-chlorobuta-1,3-diene

EC Number:

204-818-0

EC Name:

2-chlorobuta-1,3-diene

Cas Number:

126-99-8

Molecular formula:

C4H5Cl

IUPAC Name:

2-chlorobuta-1,3-diene

Constituent 2

Reference substance name:

2-Chloro-1,3-butadiene

IUPAC Name:

2-Chloro-1,3-butadiene

Constituent 3

Reference substance name:

Chloroprene

IUPAC Name:

Chloroprene

Details on test material:

Partie number - PE632
Sample number - 1130
Purity - not stated

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken for analysis from each test treatment and the control at the start of the study and at 24 hour intervals until the end of the study.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution was prepared daily,640 mg of the test substance was added to 2 L of dilution water. Undissolved particles were removed by filtration through a 7 - 12 um pore size filter paper. The pH of the stock solution was 7.9. The test media were prepared by addition of appropriate volumes of the stock solution to test vessels and dilution to 100 mL with water. Three replicates were prepared for each test treatment and control.

Algal inoculum was added to each replicate vessel resulting in a final volume of 5000 cells/mL.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG

Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).

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 – 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.

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not applicable

Test temperature:

Temperature range was 21 - 24°C

pH:

See table in the results section for details.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal exposure concentrations: 0 (control), 3.2, 10, 32, 100 and 320 mg/L. Geometric mean measured concentrations were 0.34, 0.94, 2.93, 10.7 and 38.54 mg/L.

Details on test conditions:

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 lx, was used. Light intensity was checked before start of the study.

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.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 17.3 - 22.8

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 6.0 - 9.7

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 4.8 - 8.7

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.48 - 1.6

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

All calculations were perfofmed using the statistical programme ToxRatPro Version 2.10 (released 20.02.2010). EC50 values were calcuated by probit analysis. NOEC and LOEC values were determined by multisampling comparison, using Williams Multiple Sequential t-Test.

Any other information on results incl. tables

Measured chloroprene concentrations

Test item concentration [m/L]	GC values [mg/L]
	0 h	24 h	48 h	72 h	Geometric mean
Control	3.26*	< 2.21	< 2.21	< 2.21	-
3.2	2.84	0.30	0.33	0.28	0.53
10	4.16	0.94	1.03	0.88	1.37
32	6.44	3.09	2.47	3.05	3.50
100	19.86	9.92	9.87	8.05	11.19
320	70.09	27.37	42.74	28.22	39.00

 

Comments:*The slightly elevated level of Chloroprene in the control at 0 h could not be explained, but is not relevant to the overall result of the study as all validity criteria were fulfilled.
The result of the control (0 h) was subtracted from the values of each concentration (0 h).
Values below the determination limit (2.21 mg/L) were calculated based on the mean recovery rate of the remaining concentrations at 0, 24, 48 and 72 hour.

 

pH values at the start and end of the study

Mean measured concentration [m/L]	pH values
	0 h	72 h
Control	7.9	10.4
0.34	7.8	10.5
0.94	7.8	10.5
2.93	7.7	10.5
10.7	7.7	10.2
38.54	7.7	8.0

 

 

Mean cell numbers during the study

Mean measured concentration [m/L]	Mean cell number (initial value 0.5 X 10^4cells/mL)
	24 hours	48 hours	72 hours
Control	3.0	8.0	28.0
0.53	2.0	7.0	28.0
1.37	2.0	6.0	29.0
3.50	2.0	5.0	18.0
11.19	1.0	4.0	15.0
39.00	1.0	1.0	1.0

 

  

 

Effects over the 72 hour exposure period;

Mean measured concentrations (mg/L)	72 h growth rate Mean ± CV %	% inhibition compared to the control	72 h yield Mean ± CV %	% inhibition compared to the control
Control	1.349± 0.013	-	28.2± 1.15	-
0.53	1.342± 0.012	0.6	27.5± 1.0	2.4
1.37	1.342± 0.012	0.6	27.5± 1.0	2.4
3.50	1.188± 0.011	12.0	17.2± 0.58	39.1
11.19	1.102± 0.028	18.3	13.2± 1.150	53.3
39.00	0.231± 0	82.9	0.5± 0	98.2

 

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentrations the 72 hour ErC50value was calculated to be 20.4 mg/L (with 95% confidence intervals of 17.9 – 23.3 mg/L), the corresponding no-observed effect concentration (NOEC) was 1.37 mg/L. The 72 hour EyC50value was calculated to be 8.3 mg/L (with 95% confidence intervals of 6.3 – 10.0 mg/L), the corresponding NOEC was 1.37 mg/L. 

Executive summary:

The alga Scenedesmus subspicatus were exposed to nominal chloroprene concentrations of 0 (control), 3.2, 10, 32, 100 and 320 mg/L for 72 hours according to EU Method C.3 Freshwater Alga and Cyanobacteria, Growth Inhibition test (2009).  Geometric mean measured concentrations were 0.53, 1.37, 3.50, 11.19 and 39.00 mg/L in the nominal 3.2, 10, 32, 100 and 320 mg/L treatments. Based on mean measured concentrations the 72 hour E_rC₅₀value was calculated to be 20.4 mg/L (with 95% confidence intervals of 17.9 – 23.3 mg/L), the corresponding no-observed effect concentration (NOEC) was 1.37 mg/L. The 72 hour E_yC₅₀value was calculated to be 8.3 mg/L (with 95% confidence intervals of 6.3 – 10.0 mg/L), the corresponding NOEC was 0.94 mg/L.