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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:
experimental study
Adequacy of study:
key study
Study period:
from 2017-10-16 to 2017-10-20, with the definitive exposure phase from 2017-10-17 to 2017-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
The test concentration of Disperse Blue 165 was analytically verified via HPLC-DAD in the saturated solution, the dilutions 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. At test start the measured test item concentrations were below the LOD in all loading levels. At the end of the exposure (72 hours), no analytical monitoring was carried out, as all test loadings can be considered to be below the LOD in the aged media too.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the Saturated solution
A saturated solution with a nominal loading of 100 mg test item/L was prepared once 24 hours prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of 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 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 10 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. During filtration, the filter was always kept covered.
The solution was checked via laser beam (Tyndall effect), which was positive.

Test loading
All terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The saturated solution and 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. The test loadings are based on the results of a non-GLP preliminary range finding test.

Control
Six replicates (without test item) were incubated under the same conditions as the test loadings.

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 Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.


Study design

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

Test conditions

Hardness:
Not specified
Test temperature:
min.: 22.0 max.: 23.0 mean value: 22.5
pH:
Nominal test item loading pH-values
[% of the saturated solution] Start; 0 hours End; 72 hours
100 7.76 8.11
50.0 7.80 8.18
25.0 7.85 8.62
12.5 7.90 8.92
6.25 7.94 8.98
Control 7.98 8.91
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
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 6839 cells/mL
- Control end cells density: Mean 1936653 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5911 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the saturated solution.

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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
12.5 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate und yield
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
25 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
40.8 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 38.3 - 43.2
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
24.7 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: CI: 20.8 - 29.9
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: The test media clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-10-10 to 2017-10-13. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.535 0.745 ± 0.524
95 % confidence interval 0.523 – 0.547
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95 % confidence interval 0.249 – 0.339
SD = Standard deviation
Reported statistics and error estimates:
EL-values and statistical analyses
EL-values of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and analyses
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.
The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Monotonicity of Concentration/Response was assessed by trend analysis by contrasts (significance level: 0.05).
William’s Multiple Sequential t-test on significant differences was done with a significance level of 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- ToxRat Professional, Version 3.2, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

Cell Densities

Nominal test item loading

Replicate

Cell density [cells/mL]

[% of the saturated solution]

No.

0 hours

24 hours

48 hours

72 hours

100

1

6839

2810

LOQcell density

LOQcell density

2

6839

3445

LOQcell density

2892

3

6839

3041

LOQcell density

LOQcell density

Mean

6839

3099

n.a.

n.a.

 50.0

1

6839

5856

7094

40333

2

6839

5651

5543

25741

3

6839

4952

8905

48780

Mean

6839

5486

7181

38285

 25.0

1

6839

19898

140581

1179112

2

6839

11287

67009

650032

3

6839

19418

155129

1392534

Mean

6839

16868

120906

1073893

 12.5

1

6839

27599

228384

1765716

2

6839

24764

190639

1582080

3

6839

31730

273924

1924364

Mean

6839

28031

230982

1757387

   6.25

1

6839

30955

273526

2138371

2

6839

35350

303945

2476324

3

6839

32155

326440

2273787

Mean

6839

32820

301304

2296161

Control

1

6839

32213

276359

1520693

2

6839

31804

293353

2191859

3

6839

34519

266960

1761912

4

6839

34115

314631

2026130

5

6839

34425

338004

1889252

6

6839

33135

331707

2230072

Mean

6839

33369

303502

1936653

 

LOQcell density  = Limit of quantification of the cell density (2736 cells/mL).             n.a.       = not applicable

 


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 loading

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[% of the saturated solution]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

n.a.

100

 

n.a.

100

2

 

-0.287

100

 

-3947

100

3

 

n.a.

100

 

n.a.

100

Mean

(+)

n.a.

100

(+)

n.a.

100

 50.0

1

 

0.592

69

 

33494

98

2

 

0.442

76

 

18902

99

3

 

0.655

65

 

41941

98

Mean

(+)

0.563

70

(+)

31446

98

 25.0

1

 

1.72

9

 

1172273

39

2

 

1.52

19

 

643193

67

3

 

1.77

6

 

1385695

28

Mean

(+)

1.67

11

(+)

1067054

45

 12.5

1

 

1.85

1

 

1758877

9

2

 

1.82

3

 

1575241

18

3

 

1.88

0

 

1917525

1

Mean

(-)

1.85

2

(-)

1750548

9

   6.25

1

 

1.92

-2

 

2131532

-10

2

 

1.96

-5

 

2469485

-28

3

 

1.94

-3

 

2266948

-17

Mean

(-)

1.94

-3

(-)

2289322

-19

Control

1

 

1.80

 

 

1513854

 

2

 

1.92

 

 

2185020

 

3

 

1.85

 

 

1755073

 

4

 

1.90

 

 

2019291

 

5

 

1.87

 

 

1882413

 

6

 

1.93

 

 

2223233

 

Mean

 

1.88

 

 

1929814

 

n.a. = not applicable

 


Section-by-Section and Average Specific Growth Ratesof 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

2.15

1.71

1.80

0.311

17.3

16.9

2

1.54

2.22

2.01

1.92

0.351

18.2

3

1.62

2.05

1.89

1.85

0.216

11.7

4

1.61

2.22

1.86

1.90

0.309

16.3

5

1.62

2.28

1.72

1.87

0.359

19.2

6

1.58

2.30

1.91

1.93

0.363

18.8

 

 

 

Mean

1.88

 

 

 

 

 

SD ±

0.05

 

 

 

 

CV [%]

2.57

 

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Disperse Blue 165 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item loadings): The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 40.8 (38.3 – 43.2) and 24.7 (20.8 – 29.9)% of the saturated solution, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 12.5% of the saturated solution, respectively. All effect levels are far above the water solubility limit of the substance. The substance is not considered to be harmful to freshwater algae.
Executive summary:

The toxicity of Disperse Blue 165 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3 from 2017-10-16 to 2017-10-20, with the definitive exposure phase from 2017-10-17 to 2017-10-20 at the test facility. The aim of the study was the determination of NOEL, LOEL, EL10, EL20- and EL50-values of growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 100 mg test item/L was prepared with dilution water 24±1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The saturated solution was stirred for 24 ±1hour (1100 rpm, at room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration. The following filtrate, i.e. the saturated solution was used in the test. The solution was checked via laser beam (Tyndall effect), which was positive.

 

The saturated solution and four dilution levels out of the saturated solution were tested in the definitive test in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The test media were clear throughout the test period. The environmental conditionswere within the acceptable limits.

The measured concentrations of the test item were all < LOD (0.00625 mg/L)at the start of the exposure (0 hours), therefore no analysis at the end of the exposure was necessary.

 

All effect values given are based on the nominal test item loadings of DisperseBlue 165.

NOEL, LOEL, ELx-values and 95% Confidence Intervals ofDisperseBlue 165(0 - 72 hours)

              based on the nominal test item loadings

 

Growth Rate Inhibition
nominal test item loadings [% saturated solution]

NOEL

12.5

LOEL

25.0

ErL10

24.1 (21.3 – 27.4)

ErL20

29.1 (26.4 – 32.0)

ErL50

40.8 (38.3 – 43.2)

 

Yield Inhibition

nominal test item loadings [% saturated solution]

NOEL

12.5

LOEL

25.0

EyL10

14.1 (10.9 – 17.6)

EyL20

16.5 (13.1 - 20.0)

EyL50

24.7 (20.8 – 29.9)