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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:
2015-03-15 to 2015-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Analytical monitoring:
yes
Details on sampling:
-Concentrations: The saturated solution and further four dilution levels 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 concentrations 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. The concentration levels were based on the sponsor’s proposal.

- Sampling method: Analytical evaluation of the saturated solution of Disperse Blue ANT and the control was carried out via
HPLC-UV from freshly prepared media after 0 hours. Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. The recovery rates of the test item at the start were below the limit of quantification. Therefore, no analytical evaluation after 96 h was carried out.


The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
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) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.2 µm, RC, MACHEREY-NAGEL).
Application Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
- Differential loading: The saturated solution and further four dilution levels of the
(nominal) 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 concentrations 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. The concentration levels were based on the sponsor’s proposal.
- Dilution water: Threefold concentrated AAP medium according to the guidelines, composition of dilution water see below -Controls: Eight replicates (without test item) were tested under the same test conditions as the test replicates.
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, standard growth medium
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
Not measured
Test temperature:
Mean: 24.8 °C, Min: 24.5 °C, Max:25.6 °C
pH:
Nomina
test item concentration pH-value
[mg/L] Start; 0 h End; 96 h
5.00 7.94 9.86
2.50 7.86 9.87
1.25 7.71 9.84
0.625 7.62 9.95
0.313 7.58 9.90
Control 7.55 9.67
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 6289107 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: According to the guideline (OECD)

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
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), 40.7 µE x m-2 x s-1 (measured, n = 32, CV = 6.69%)



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 definitive test at the nominal concentration level of 100 % of the saturated solution.

- Microscopic evaluation : Microscopic evaluation of the cells was carried out daily. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.


TEST CONCENTRATIONS
- Range finding study: No












Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
96 h
Dose descriptor:
EC20
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, yield
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate, 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 concentration 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:
Biomass inhibition 0.284 (CI 0.236 - 0.327)
Rate-related inhibition: 0.749 (CI 0.715 - 0.788) mg/L
Yield Inhibition: 0.282 (CI 0.239 - 0.318) mg/L
Reported statistics and error estimates:
EC-values and statistical analysis:

EC-values and statistical analyses: As no effects of the test item on biomass, growth rate and yield were observed, EC10-, EC20- and EC50-values were deduced directly from the results.

NOEC, LOEC and statistical analyses:The NOEC / LOEC was determined by calculation of statistically significant differences of biomass integrals, growth rate and yield between test substance treated and control groups. As a 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 tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.
t.



          

  Cell Densities (nominal test item concentration)

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

58411

498743

2591246

6866931

2

10010

59681

561087

3191225

7165227

3

10010

66458

539104

3354673

7782016

4

10010

65295

573343

2990709

6377464

Mean

10010

62461

543069

3031963

7047910

2.50

1

10010

50229

523141

2356736

6188928

2

10010

49204

603310

3236070

6996761

3

10010

62730

572025

2763537

7334571

4

10010

67565

589374

4046977

8614812

Mean

10010

57432

571963

3100830

7283768

1.25

1

10010

46783

472652

2336163

5794042

2

10010

44553

469641

2439526

6148536

3

10010

62068

586790

3186521

6651550

4

10010

59481

587103

3000117

7190942

Mean

10010

53221

529047

2740582

6446268

0.625

1

10010

53880

503360

2584346

7042798

2

10010

43434

491179

2337355

6235466

3

10010

66185

569793

2803678

7103887

4

10010

57311

599409

2672593

6870819

Mean

10010

55203

540935

2599493

6813243

0.313

1

10010

57279

504978

2507389

6268331

2

10010

55329

501603

2352282

6460631

3

10010

44923

435057

2283918

4154792

4

10010

59531

483063

2516170

6373575

Mean

10010

54266

481175

2414940

5814332

Control

1

10010

52459

485509

2459596

6362160

2

10010

40110

373943

1798653

5739601

3

10010

56442

473919

2251868

6176634

4

10010

42841

452707

2285925

6159825

5

10010

63457

580907

2638160

6331929

6

10010

49972

474834

3377002

6872074

7

10010

45093

454902

2355356

5934410

8

10010

41596

458502

2746791

6736222

Mean

10010

48996

469403

2489169

6289107

 

Section-by-Section and Average Specific Growth Rates of the Control Group
(0 - 96 hours)

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 96 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

72 - 96 hours

Control

1

1.66

2.23

1.62

0.950

1.61

0.521

32.3

34.5

2

1.39

2.23

1.57

1.16

1.59

0.461

29.1

3

1.73

2.13

1.56

1.01

1.61

0.464

28.9

4

1.45

2.36

1.62

0.991

1.61

0.568

35.3

5

1.85

2.21

1.51

0.876

1.61

0.569

35.3

6

1.61

2.25

1.96

0.710

1.63

0.669

41.0

7

1.51

2.31

1.64

0.924

1.60

0.570

35.7

8

1.42

2.40

1.79

0.897

1.63

0.632

38.8

 

 

 

Mean

1.61

 

 

 

 

 

SD ±

0.02

 

 

 

 

CV [%]

0.93

 

SD = Standard deviation CV = Coefficient of variation

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

1.66

2.23

1.62

1.83

0.339

18.5

21.5

2

1.39

2.23

1.57

1.73

0.444

25.7

3

1.73

2.13

1.56

1.81

0.292

16.2

4

1.45

2.36

1.62

1.81

0.481

26.6

5

1.85

2.21

1.51

1.86

0.351

18.9

6

1.61

2.25

1.96

1.94

0.322

16.6

7

1.51

2.31

1.64

1.82

0.431

23.7

8

1.42

2.40

1.79

1.87

0.493

26.3

 

 

 

Mean

1.83

 

 

 

 

 

SD ±

0.06

 

 

 

 

CV [%]

3.30

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:
yes
Conclusions:
In this study, Disperse Blue ANT was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata with the following effect values (nominal test item loading rates): The EL50-values for inhibition of biomass (EbL50), growth rate (ErL50) and yield (EyL50) after 72 and 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 ANT (batch number:EAG-149-1.0.0) 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 (2009) and EPA Guideline 712-C-006: OCSPP 850.4500 (2012) atDr.U.Noack-Laboratorienin 31157 Sarstedt, Germanyfrom2015-03-15 to 2015-03-20with the definitive exposure phase from2015-03-16 to 2015-03-20.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 solution1and 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 rates 0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L. Four replicates were tested for each test itemloading rateand eight replicates for the control. The environmental conditionswere within the acceptable limits.

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

The concentrations of the test itemDisperse Blue ANTin the saturated solution and the controlwere analytically verified via HPLC-UV at the start of exposure. The recovery rates ofthe test item at the start were below the limit of quantification.

All effect values were based on the nominalloading ratesof the test itemDisperse Blue ANT.

NOEL, LOEL, EL-Values of Disperse Blue ANT

based on nominal test itemloading 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 

Nominaltest 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

Nominaltest 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

 

Description of key information

No adverse effects noted at the limit of solubility and far beyond (OECD 201, structural analogue substance).

Key value for chemical safety assessment

Additional information