2,3-dichlorobuta-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:

2010-04-20 till 2010-05-27

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:

2,3-dichloro-1,3-butadiene

IUPAC Name:

2,3-dichloro-1,3-butadiene

Constituent 2

Reference substance name:

2,3-dichlorobuta-1,3-diene

EC Number:

216-721-0

EC Name:

2,3-dichlorobuta-1,3-diene

Cas Number:

1653-19-6

Molecular formula:

C4H4Cl2

IUPAC Name:

2,3-dichlorobuta-1,3-diene

Constituent 3

Reference substance name:

2,3-dichlorobutadiene-1,3

IUPAC Name:

2,3-dichlorobutadiene-1,3

Details on test material:

Partie number - Q101
Sample number - 1152
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,271.2 uL 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 8.0. 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. Test vessels were filled to reduce loss of test material due to volatisation.

A volume of 0.667 mL of 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), 5.0, 10, 20, 40, 80 and 160 mg/L. Geometric mean measured concentrations were 0.422, 0.888, 1.464, 2.869, 5.855 and 17.182mg/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.:

4.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 3.8 - 5.0

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1.7 - 2.9

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

<= 0.422 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.:

2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 2.7 - 3.2

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 1.3 - 1.9

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.422 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

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 (relaeased 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 2,3-dichloro-1,3-butadiene concentrations

Test item concentration [m/L]	GC values [mg/L]
	0 h	24 h	48 h	72 h	Geometric mean
Control	< 0.629	< 0.629	< 0.629	< 0.629	-
5.0	0.793	0.399*	0.405*	0.247*	0.422
10	1.578	0.869	0.917	0.495*	0.888
20	2.694	1.406	1.398	0.868	1.462
40	5.199	2.765	2.900	1.625	2.869
80	10.562	5.540	5.872	3.420	5.855
160	29.598	16.587	15.603	11.378	17.182

*Values below the limit of quantification (0.629 mg/L) were calculated on basis of the mean recovery rate of the remaining concentrations at 24, 48 and 72 hour

pH values at start and end of the study

Test item concentration [mg/L]	pH values at 0 h and 72 h
	0 h	72 h
Control	7.5	10.4
5.0	7.7	10.5
10	7.8	10.5
20	7.8	10.5
40	7.8	9.4
80	7.8	8.0
160	7.8	7.9

Cell numbers during the study

Mean measured concentrations (mg/L)	Mean cell number (initial value 0.5 X 10^4cells/mL)
	24 hours	48 hours	72 hours
Control	2.0	7.7	20.0
0.422	2.0	8.0	19.0
0.888	2.0	7.7	18.0
1.464	1.7	6.3	18.7
2.869	1.0	3.0	10.7
5.855	1.0	1.0	1.3
17.182	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	1.229 ±0.03	-	19.5 ± 2.0	-
0.422	1.212 ±0.02	1.3	18.5 ± 1.0	5.1
0.888	1.195 ±0	2.8	17.5 ± 0	10
1.464	1.206 ±0.03	1.8	18.2 ±1.5	6.8
2.869	1.202 ± 0.02	17	10.2 ± 0.58	48
5.855	0.308 ± 0.13	75	0.83 ± 0.58	96
17.182	0.231 ±0	81	0.5 ± 0	97

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentrations the 72 hour ErC50 value for Scenedesmus subspicatus exposed to 2,3-dichlorobutadiene-1,3 was calculated to be 4.4 mg/L (with 95% confidence intervals of 3.8 - 5.0 mg/L), the corresponding no-observed effect concentration (NOEC) was 0.888 mg/L. The 72 hour EyC50 value was calculated to be 2.9 mg/L (with 95% confidence intervals of 2.7 – 3.2 mg/L), the corresponding NOEC was 0.422 mg/L.

Executive summary:

The alga Scenedesmus subspicatus were exposed to nominal 2,3-dichlorobutadiene-1,3 concentrations of 0 (control), 5.0, 10, 20, 40, 80 and 160 mg/L for 72 hours according to EU Method C.3 Freshwater Alga and Cyanobacteria, Growth Inhibition test (2009).  To reduce volatilisation of 2,3-dichlorobutadiene-1,3 the study was conducted in sealed test vessels.

Geometric mean measured concentrations were 0.422, 0.888, 1.464, 2.869, 5.855 and 17.182 mg/L in the nominal 5.0, 10, 20, 40, 80 and 160 mg/L treatments. Based on mean measured concentrations the 72 hour E_rC₅₀value was calculated to be 4.4 mg/L (with 95% confidence intervals of 3.8 - 5.0 mg/L), the corresponding no-observed effect concentration (NOEC) was0.888 mg/L. The 72 hour E_yC₅₀value was calculated to be 2.9 mg/L (with 95% confidence intervals of 2.7 – 3.2 mg/L), the corresponding NOEC was 0.422 mg/L.