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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:
2015-03-08 to 2015-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test item: Disperse Blue 1092

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to the nominal loading rates 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. The loading levels are based on a proposal of the sponsor.


- Sampling method: The test concentrations of Disperse Blue 1092 were analytically verified via HPLC-UV in the saturated solution and the control at the start of the exposure (0 hours). At the start of the exposure (0 hours), sampling was carried out after preparation of the loading levels.
As the measured test item concentration was below the LOQ in the saturated solution, the test item concentrations at the other loading levels were considered to be also below the LOQ. At the end of the exposure (96 hours), no analytical monitoring was carried out, because a measured test item concentration above the LOQ was not observed at the start of the exposure.
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The saturated solution with a nominal loading of 5.00 mg/L was prepared once with dilution water. The saturated solution was stirred for 24 hours (1100 rpm) at room temperature with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.2 µm, RC, MACHEREY-NAGEL).

- Differential loading: The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to the nominal loading rates 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. The loading levels are based on a proposal of the sponsor.

- Dilution water: Threefold concentrated AAP medium according to the guidelines
-Controls: Eight replicates (without test item) were tested under the same test conditions as the test item groups.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 hours per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, Nutrient medium Z according to LÜTTGE et al. (1994)
- Any deformed or abnormal cells observed: No

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 24.4 °C, Min: 23.8°C, Max: 25.0 °C
pH:
Nominal test item
loading rate pH-value
[mg/L] Start; 0 hours End; 96 hours
5.00 7.50 10.1
2.50 7.43 10.1
1.25 7.42 10.1
0.625 7.43 10.1
0.313 7.44 9.91
Control 7.46 9.96

Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to information on materials and methods
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks (sterile)
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Agitation: Test containers were placed on a rotary shaker and oscillated at appr. 100 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: 10010 cells/mL
- Control end cells density: Mean 5142808 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: Yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Threefold concentrated AAP medium according to the guidelines

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NaNO3 76.5
MgCl2  6 H2O 36.492
CaCl2  2 H2O 13.23
MgSO4  7 H2O 44.1
K2HPO4 3.132
NaHCO3 45
H3BO3 0.5565
MnCl2  4 H2O 1.246
ZnCl2 9.81 x 10-3
CoCl2  6 H2O 4.29 x 10-3
CuCl2  2 H2O 0.036 x 10-3
NaMoO4  2 H2O 21.78 x 10-3
FeCl3  6 H2O 0.4794
Na2-EDTA 0.9
pH 7.5  0.1

The pH of the test medium has to be 7.5  0.1 and was adjusted prior to testing with addition of 1 M NaOH and HCl.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality:35 - 45 µE x m-2 x s-1 (nominal), mean: 42.4 µE x m-2 x s-1, measured values: 37.2 - 44.9 µE x m-2 x s-1 (measured, n = 31, CV = 5.19%)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the definitive test at the nominal loading level of 5.00 mg/L.


TEST CONCENTRATIONS
- Range finding study: None











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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
96 h
Dose descriptor:
LOELR
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
96 h
Dose descriptor:
EL10
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
96 h
Dose descriptor:
EL20
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibition of Yield
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass, Growth Rate, Inhibtion of Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: Microscopic evaluation of the cells at the start of the incubation period, after 24, 48, 72 hours and at test end revealed no morphological abnormalities in any test item loading rate or control.
- 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
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Current Study Valid Range (average ± 3 x SD)
Biomass inhibition
EbC50 0.284 0.442 ± 0.278
95 %
confidence interval 0.236 – 0.327
Growth Rate inhibition
ErC50 0.749 0.821 ± 0.388
95 %
confidence interval 0.715 - 0.788
Yield inhibition
EyC50 0.282 0.429 ± 0.261
95 %
confidence interval 0.239 - 0.318


Reported statistics and error estimates:
EC-values and statistical analysis:

As no effects of the test item on biomass, growth rate and yield were observed, EL10-, EL20- and EL50-values were deduced directly from the results.



NOEC/LOEC values:
The NOEL / LOEL was determined by calculation of statistical
significance of biomass integrals, growth rate and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL 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 tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.




Any other information on results incl. tables

Cell Densities (Nominal test item loading rates)

Nominal test item loading rate

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

96 hours

5.00

1

10010

60729

424426

2080159

6213219

2

10010

60130

442904

2176121

5979524

3

10010

66499

514743

2481316

7757511

4

10010

61287

506401

2448953

6864880

Mean

10010

62161

472119

2296637

6703784

2.50

1

10010

48661

371165

1905609

5507619

2

10010

51199

350806

1850604

5482656

3

10010

54429

379117

1990219

6246962

4

10010

54940

423799

2201146

6806550

Mean

10010

52307

381222

1986895

6010947

1.25

1

10010

56332

414090

1801745

5088273

2

10010

45303

296728

1620422

5380925

3

10010

55705

397959

1866221

5889960

4

10010

56586

403591

2067929

6594055

Mean

10010

53482

378092

1839079

5738303

0.625

1

10010

53488

430786

1841447

4929466

2

10010

57420

441022

2020512

5630048

3

10010

65775

487234

1996365

6245332

4

10010

54501

459688

2207167

6739063

Mean

10010

57796

454683

2016373

5885977

0.313

1

10010

52732

432668

1919282

5113737

2

10010

50728

384373

1731499

5366374

3

10010

64069

502425

2215446

6236175

4

10010

57333

489015

2197383

7210592

Mean

10010

56216

452120

2015903

5981720

Control

1

10010

47125

447394

1586804

4618500

2

10010

49994

386192

1708355

4590151

3

10010

62403

488677

2029732

5471492

4

10010

62378

460378

1988713

5005357

5

10010

60704

597521

2054632

4908643

6

10010

80934

627903

2716265

6182110

7

10010

69742

492979

2306767

4908016

8

10010

73499

485265

2415586

5458196

Mean

10010

63347

498289

2100857

5142808

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 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.55

2.25

1.27

1.69

0.507

30.0

19.1

2

1.61

2.04

1.49

1.71

0.293

17.1

3

1.83

2.06

1.42

1.77

0.321

18.1

4

1.83

2.00

1.46

1.76

0.274

15.5

5

1.80

2.29

1.24

1.78

0.526

29.7

6

2.09

2.05

1.47

1.87

0.350

18.7

7

1.94

1.96

1.54

1.81

0.234

12.9

8

1.99

1.89

1.61

1.83

0.201

11.0

 

 

 

Mean

1.78

 

 

 

 

 

SD ±

0.06

 

 

 

 

CV [%]

3.32

 

Microscopic evaluation of the cells at the start of the incubation period, after 24, 48, 72 hours and at test end revealed no morphological abnormalities in any test item loading rate or control. The environmental conditions (pH-value, water temperature, light intensity) were within the acceptable limits

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study Disperse Blue 1092 was found to not inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata with the following effect values (nominal test item loading rates): The EL50-values with 95 % confidence intervals for inhibition of biomass (EbL50), growth rate (ErL50) and yield (EyL50) after 72 hours were > 5.00 mg/L. The EL50-values with 95 % confidence intervals for inhibition of biomass (EbL50), growth rate (ErL50) and yield (EyL50) after 96 hours were > 5.00 mg/L. The NOEL-values for inhibition of biomass, growth rate and yield after 72 and 96 hours were 5.00 mg/L.
Executive summary:

The toxicity of Disperse Blue 1092 (batch number:EAG-148-1.0.0) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201, Commission Regulation (EC) No. 761/2009, C.3 and EPA Guideline 712-C-006: OCSPP 850.4500, 2012 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2015-03-08 to 2015-03-13 with the definitive exposure phase from 2015-03-09 to 2015-03-13. The aim of the study was to assess the effects on biomass integrals, growth rate and yield over a period of 96 hours.

The study was conducted under static conditions with an initial cell density of 10010 cells/mL. The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0-100 % of the saturated solution, corresponding to the nominal loading levels 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. Four replicates were tested for each loading level and eight replicates for the control. The environmental conditionswere within the acceptable limits.

No recovery phase was performed, because no loading level was inhibited by more than 50%.

The concentrations of the test item Disperse Blue 1092 were analytically verified via HPLC-UV at the start of the saturated solution and the control. The recovery rate of the saturated solution at the start was below the limit of quantification.

All effect values were based on the nominal loading levels of the test item Disperse Blue 1092. The results are summarized in the following table:

 

NOEL, LOEL, EL-Values and 95 % Confidence Intervals of Disperse Blue 1092

    based on nominal test item loading rates

 

 

Inhibition of Biomass Integrals

Nominal test item loading rates [mg/L]

0 - 72 hours

0 - 96 hours

NOEL

5.00

5.00

LOEL

> 5.00

> 5.00

EbL10

> 5.00

> 5.00

EbL20

> 5.00

> 5.00

EbL50

> 5.00

> 5.00

 

Inhibition of Growth Rate

Nominal test item loading rates [mg/L]

NOEL

5.00

5.00

LOEL

> 5.00

> 5.00

ErL10

> 5.00

> 5.00

ErL20

> 5.00

> 5.00

ErL50

> 5.00

> 5.00

 

Inhibition of Yield

Nominal test item loading rates [mg/L]

NOEL

5.00

5.00

LOEL

> 5.00

> 5.00

EyL10

> 5.00

> 5.00

EyL20

> 5.00

> 5.00

EyL50

> 5.00

> 5.00