Registration Dossier

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:
from 2017-07-16 to 2017-07-20, with the definitive exposure phase from 2017-07-17 to 2017-07-20
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 284 was analytically verified via HPLC-DAD 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 level. At the end of the exposure (72 hours), no analytical monitoring was carried out, because a measured test item concentration above LOD was not observed at the start of the exposure.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the Saturated solution
A saturated solution with a nominal loading of 10.0 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 will always be kept covered.
The solution was checked via laser beam for undissolved test item (Tyndall effect), which was negative.

Test loading
All terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. One limit concentration were tested with a nominal loading of 10.0 mg/L.

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): 3 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:
yes
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
[mg/L] Start; 0 hours End; 72 hours
10.0 7.80 8.54
Control 7.91 8.75
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Nominal: 100% of the saturated solution
Measured: < LOQ
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 1914404 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:
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: 5771 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.Self-fluorescence was observed in the range finding test. A separate replicate for the loading levels were prepared without algae and treated the same as the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.

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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EL10/20/50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
other: EL10/20/50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
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 saturated solution was visually clear orange throughout the exposure period. The Tyndall effect negative at the saturated solution.
- 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 2017-04-18 to 2017-04-21. 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 0.559 0.754 ± 0.518
95% confidence interval 0.538 – 0.581
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95% confidence interval 0.242 – 0.341
SD = Standard deviation
Reported statistics and error estimates:
EL-values and statistical analyses
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the single treatment level (Limit test design).

NOEL and LOEL values
NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOEL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. 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.

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.

Any other information on results incl. tables

Cell Densities

 

Nominal test item loading

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

10.0

1

6839

24164

199680

1623355

2

6839

24553

244062

1874230

3

6839

28418

233926

1823551

4

6839

28067

317141

2078230

5

6839

27236

281819

2078523

6

6839

28755

285213

2216221

Mean

6839

26866

260307

1949018

Control

1

6839

29477

289637

2021642

2

6839

35584

296296

2274155

3

6839

35084

318803

2065707

4

6839

26818

248006

1828409

5

6839

21039

232404

1709203

6

6839

25404

174996

1587306

Mean

6839

28901

260024

1914404

 


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

Growth rate inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

10.0

1

 

1.82

3

 

1616516

15

2

                                                                                        

1.87

0

 

1867391

2

3

 

1.86

1

 

1816712

5

4

 

1.91

-2

 

2071391

-9

5

 

1.91

-2

 

2071684

-9

6

 

1.93

-3

 

2209382

-16

Mean

(-)

1.88

0

(-)

1942179

-2

Control

1

 

1.90

 

 

2014803

 

2

 

1.94

 

 

2267316

 

3

 

1.90

 

 

2058868

 

4

 

1.86

 

 

1821570

 

5

 

1.84

 

 

1702364

 

6

 

1.82

 

 

1580467

 

Mean

 

1.88

 

 

1907565

 

 

 

 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

1.46

2.29

1.94

1.90

0.414

21.8

22.4

2

1.65

2.12

2.04

1.94

0.251

13.0

3

1.64

2.21

1.87

1.90

0.288

15.1

4

1.37

2.22

2.00

1.86

0.445

23.9

5

1.12

2.40

2.00

1.84

0.653

35.5

6

1.31

1.93

2.21

1.82

0.457

25.2

 

 

 

Mean

1.87

 

 

 

 

 

SD ±

0.04

 

 

 

 

CV [%]

2.36

 

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Disperse Blue 284 was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. Effect values were determined as follows (based on nominal test item loadings): The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 10.0 mg/L, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 10.0 mg/L, respectively. No effects could be determined up to the water solubility limit of the test item and beyond.
Executive summary:

The toxicity of Disperse Blue 284 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016/Method C.3 from 2017-07-16 to 2017-07-20, with the definitive exposure phase from 2017-07-17 to 2017-07-20 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 10.0 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, 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 negative.

Six replicates were tested for the limit concentration and six for the control. The test media were clear and slightly blue coloured throughout the test period. The environmental conditions were within the acceptable limits.

The measured concentration of the test item was even < LOD 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 284.

 

NOEL, LOEL, ELx-values ofDisperseBlue 284(0 - 72 hours)

                  based on the nominal test item loadings

 

Growth Rate Inhibition
nominal test item loadings [mg/L]

NOEL

10.0

LOEL

> 10.0

ErL10

> 10.0

ErL20

> 10.0

ErL50

> 10.0

 

Yield Inhibition

nominal test item loadings [mg/L]

NOEL

10.0

LOEL

> 10.0

EyL10

> 10.0

EyL20

> 10.0

EyL50

> 10.0