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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04. May 2010 - 15. May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.25 (recovery values below LOQ), 12.5 (11.1), 25 (16.6), 50 (40.6), 100 (38.6) mg/L nominal (measured)
- Sampling method: samples drawn from additional beakers without algae, kept under the same experimental conditions
- Sample storage conditions before analysis: samples were drawn, immediately transferred to the analytical laboratory and analyzed on the same day
Vehicle:
no
Details on test solutions:
Six 2 L beakers were prepared. One was filled with 1.5 L of culture medium for the control. The following preparations of Dodecanedioic acid were used: 6.25 mg + 1000 mL culture medium; 12.5 mg + 1000 mL culture medium; 25 mg + 1000 mL culture medium; 50 mg + 1000 mL culture medium; 100 mg + 1000 mL culture medium. The volumes of culture medium were weighed into five 2L beakers each, stirred with magnetic stirrer (300 rpm) and the corresponding amounts of the test substance were added to the stirring medium. Preparations were stirred for 24h.
Afterwards the test substance preparations were filtered through a filter paper circle Ø 320 mm (Whatman Schleicher & Schuell, Dassel, Germany). The control was treated the same way.To avoid inhibition of algae growth induced by the low pH value of the test substance (as shown in the NON-GLP pre-test: test substance preparation of 100 mg/L = pH 5.6, complete growth inhibition), the pH value of test concentrations was adjusted to 8.1 with 0.1N NaOH after filtration.
For the control group, 6 test vessels (300 mL) were filled with 150 mL culture medium and were inoculated with 7.5 x 105 cells (final concentration 5 x 103 cells per mL) of algal suspension (pre-culture) according to the calculation of the concentration from the measurement with the Coulter Counter. Three test vessels were filled each with 150 mL of the prepared test concentrations; the remaining solution was used to determine the pH-value at the beginning of the test and for analytical determinations. Afterwards the appropriate volume (containing 7.5 x 105 cells) of the algae pre-culture was added to each of the three test vessels.
Five additional samples in 300 mL conical flasks were prepared for analytical determinations at the test site AQura GmbH, Marl, Germany. 300 mL test solution from the concentration levels 12.5; 25; 50; 100 mg* test substance/L culture medium and pure culture medium (control, only 0h) were filled each into the conical flasks and stored under the same conditions as the solutions in the test vessels. After 0 h, 24 h, 48 h and 72 h, 30 mL of each sample were transferred to a 50 mL glass flask, which was closed with screw cap and was immediately shipped to the test site and analytical determination was performed on the same day.

*6.25 mg/L was excluded from analysis because this nominal concentration results in recovery below the limit of quantification (LOQ: 5.0 mg/L) as shown in a NON-GLP pre-test at the test site AQura GmbH.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81
- Source (laboratory, culture collection): Collection of Algae Cultures, Institute for Plant Physiology, University of Göttingen, Germany
- Age of inoculum (at test initiation): < 2 weeks
- Method of cultivation: A slant agar tube with Desmodesmus subspicatus is stored in the refrigerator. Two 300 mL conical flasks, filled with 100 mL of sterile culture medium, were inoculated with algae from the slant agar. Twice a week, the algae stock culture was transferred: 1 mL of the stock culture was diluted in two 300 mL conical flasks with 100 mL of fresh sterile culture medium. After two weeks, these stock cultures were discarded and new cultures started as described above.

ACCLIMATION
- Acclimation period: none
- Culturing media and conditions (same as test or not): not
- Any deformed or abnormal cells observed: no such cells observed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
25.2 - 25.4
pH:
7.65 - 9.75
Nominal and measured concentrations:
6.25 (-*), 12.5 (11.1), 25 (16.6), 50 (40.6), 100 (38.6) mg/L nominal concentrations (measured concentrations)
* recovery values for 6.25 mg/L were below limit of quantification of the analytical method used.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glas, 300 mL volume, filled with 150 mL culture medium
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): static test
- Initial cells density: 5000 cells /mL
- Control end cells density: 1268333 cells /mL
- No. of vessels per concentration (replicates): 3 (3 for each measuring time point 0, 24, 48, 72 h respectively)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: -

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark
- Light intensity and quality: lighting supplied by fluorescent tubes Osram Dulux L, "Cool White" was 0.72 x 1020 photons per m2s = 8000 +/- 1000 Lux
- Salinity (for marine algae): -

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
- Chlorophyll measurement: -

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none, limit test
- Justification for using less concentrations than requested by guideline: limit test as described by guideline
- Range finding study: 0.1, 1, 10, 100 mg/L nominal
- Test concentrations: 6.25, 12.5, 25, 50, 100 mg/L nominal
- Results used to determine the conditions for the definitive study: growth inhibition observed in pre test
Reference substance (positive control):
yes
Remarks:
potassium dichromate was tested in an independent experiment in May 2010 and adequate sensitivity of the algal culture was demonstratedpotassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 38.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
11.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other:
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium:
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.9 mg/L
- Other:
Reported statistics and error estimates:
Statistical analysis was done with ToxRat Professional (ToxRat Solutions GmbH, Alsfeld, Germany)

Table: EC (probit analysis using linear max. likelihood regression), LOEC and NOEC values (Welch t-test for Inhomogeneous Variances with Bonferroni-Holm Adjustment/Growth rate) and for actual concentrations (Williams Multiple Sequential t-test Procedure/Yield) and (t-test for Inhomogeneous Variances with Bonferroni-Holm Adjustment/Growth rate), determined after 72 hours exposure ofDesmodesmus subspicatusto different concentrations of test substance Dodecanedioic acid

 

 

Effective concentrations (mg/L)

 

nominal concentrations

actual concentrations

Yield

Growth rate

 

Yield

Growth rate

 

EC10       (0-72h)

10.18

11.56

7.43

9.95

EC20       (0-72h

12.58

28.94

9.39

19.47

EC50       (0-72h)

18.83

>100*

14.72

>38.6*

95 % Confidence interval

15.66 – 22.53

107.66 – 411.18

10.99 – 19.59

50.93 – 152.90

LOEC    (0-72h)

n.d.

25.0

11.1

16.6

NOEC    (0-72h)

n.d.

12.5

<11.1

11.1

 

n. d.not determined due to mathematical reasons or inappropriate data

 

*As EC50values, calculated by the statistic software ToxRat® Professional, were above the highest test concentration, they were presented as >100 mg/L and >38.6 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The ErC50 (72 h), based on nominal concentrations, was determined as >100 mg/L, the ErC50 (72 h) based on measured concentrations was determined as > 38.6 mg/L, indicating that the test substance does not pose a significant hazard to algae.
Executive summary:

The test substance dodecanedioic acid was tested in the Alga, Growth Inhibition Test according to the OECD Guideline for the Testing of Chemicals No. 201 (23 March 2006) using the single cell green algaeDesmodesmus subspicatusas test organism. The experiment was carried out as a static test, i.e. the test substance preparations were not exchanged for the whole exposure period of 72 hours. Based on non-GLP pre-test results the main test was performed with test substance concentrations of nominal 6.25, 12.5, 25, 50 and 100 mg/L culture medium. During this study the test substance concentration of nominal 12.5, 25, 50 and 100mg/L in culture medium and the control (culture medium) were determined by the test site AQura GmbH by HPLC. Recovery rates of the test substance preparation 6.25 mg/L were below the limit of quantification and thus excluded from the analysis. The results of this study show that the test substance dodecanedioic acid induced inhibition of algal growth from 25 mg/L (nominal) / 16.6 mg/L (measured) upward with an EC50 (72h) for growth rate of > 100 mg/L (nominal) / > 38.6 mg/L (measured) and an NOEC for growth rate of 12.5 mg/L (nominal) / 11.1 mg/L (measured).

Description of key information

The toxicity of dodecanedioic acid on the alga Desmodesmus subspicatus was tested over a period of 72 hours according to OECD TG 201. Five concentrations ranging from 6.25 to 100 mg/L (nominal) were tested. The results of this study show that the test substance dodecanedioic acid induced inhibition of algal growth from 16.6 mg/L (measured) upwards with an EC50 (72h) for growth rate of > 38.6 mg/L (geometric mean) and a NOEC of 11.1 mg/L (measured).

Key value for chemical safety assessment

EC50 for freshwater algae:
38.6 mg/L
EC10 or NOEC for freshwater algae:
11.1 mg/L

Additional information

The ErC50 (72 h) based on measured concentrations was determined as > 38.6 mg/L, indicating that the test substance does not pose a significant hazard to algae.