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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2011-10-28 to 2011-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guidelines and compliant to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
according to German Chemikaliengesetzt and directive 88/320/EEC

Test material

Constituent 1
Reference substance name:
Dimethyl 5-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azoterephthalate
EC Number:
249-955-7
EC Name:
Dimethyl 5-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azoterephthalate
Cas Number:
29920-31-8
IUPAC Name:
dimethyl 5-({2-oxo-1-[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)carbamoyl]propyl}diazenyl)isophthalate

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The limit test item concentration and control were analytically verified via HPLC-DAD after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from additional replicates without algae. After 72 hours samples were taken directly from pooled test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 C° until analysis, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/L was freshly prepared with dilution water. The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Centrifugation for 10 min at 10000 rpm was found to be insufficient.
- Eluate: Dilution water
- Differential loading: Saturated solution* corresponding to a nominal test item concentration of 1.00 mg/L, six replicates
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
Dilution water: 0.24 mmol Ca+mg/L.
Test temperature:
min.: 21.5 max.: 22.5, mean value: 22.0
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.86 8.32
Control 7.92 8.72
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: Mean 876660 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 69.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)

Nominal
test item concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 -1 -3
10 2 11
1 3 14


Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Due to very poor solubility of test item, concentrations in water were below LOQ of analytical method (0.1 mg/L)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Due to very poor solubility of test item, concentrations in water were below LOQ of analytical method (0.1 mg/L)
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: 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: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328


Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Any other information on results incl. tables

NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on nominal concentration [mg/L]

                               

Rate-related Inhibition

NOEC

1.00

LOEC

> 1.00

ErC10

> 1.00

ErC20

> 1.00

ErC50

> 1.00

Inhibition of Yield

NOEC

1.00

LOEC

> 1.00

EyC10

> 1.00

EyC20

> 1.00

EyC50

> 1.00

Cell Densities

 

Nominal
test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

1.00

1

4910

24018

175395

899328

 

2

4910

24065

170213

829699

 

3

4910

28137

174989

949731

 

4

4910

22779

156551

815888

 

5

4910

27027

172230

820796

 

6

4910

26983

145096

824520

 

Mean

4910

25502

165746

856660

 

Control

1

4910

26330

176776

968484

 

2

4910

26404

174874

908298

 

3

4910

26699

176620

887413

 

4

4910

22674

191186

822996

 

5

4910

25392

166953

756650

 

6

4910

29583

169072

916118

 

Mean

4910

26180

175914

876660

 

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal
test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

1.00

1

1.74

-0.55

894418

-2.60

2

1.71

 1.00

824789

 5.39

3

1.76

-1.61

944821

-8.38

4

1.70

 1.33

810978

 6.97

5

1.71

 1.21

815886

 6.41

6

1.71

 1.12

819610

 5.98

Mean

(-)

1.72

  0.420

(-)

851750

 2.30

Control

1

1.76

963574

2

1.74

903388

3

1.73

882503

4

1.71

818086

5

1.68

751740

6

1.74

911208

Mean

1.73

871750

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.68

1.90

1.70

1.76

0.124

    7.04

9.78

2

1.68

1.89

1.65

1.74

0.131

    7.55

3

1.69

1.89

1.61

1.73

0.142

    8.17

4

1.53

2.13

1.46

1.71

0.370

21.6

5

1.64

1.88

1.51

1.68

0.189

11.2

6

1.80

1.74

1.69

1.74

0.053

    3.04

Mean

1.73

 

SD ±

0.03

 

CV [%]

1.70

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L. All effect levels are given based on the nominal concentration of the test item, which has been analytically verified by HPLC-DAD.
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5 x 103 cells/mL. Based on a preliminary range finding the saturated solution* was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The saturated solution* and the control were analysed by HPLC-DAD after 0 h (fresh media) and 72 h (old media).

The concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution* and in the old media (72 h) was< LOQ. All effect values of the test item are given based on the nominal concentration.

Table 1:   NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on nominal concentration [mg/L]

                               

Rate-related Inhibition

NOEC

1.00

LOEC

> 1.00

ErC10

> 1.00

ErC20

> 1.00

ErC50

> 1.00

Inhibition of Yield

NOEC

1.00

LOEC

> 1.00

EyC10

> 1.00

EyC20

> 1.00

EyC50

> 1.00