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Ecotoxicological information

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:
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
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 concentrationsopen allclose all
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
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
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
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
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
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 104cells/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 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 0.94 mg/L.