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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:
2017-11-17 to 2017-12-07
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch number
3. April 2010

CAS number
49744-28-7

Sum formula
C17H13N3O4

Molecular weight
323.31 g/mol

Purity of main component (certified)
98.50% (w/w) (comp. 1)


Water solubility
3.3 µg/L

Appearance
Red powder

pH value
9.1 (10% H2O)

Stability
Stable in water and light at room temperature

Expiry date
2020-04-03

Recommended storage
Room temperature

Sampling and analysis

Analytical monitoring:
no
Remarks:
No analytical monitoring was carried out due to the low solubility of the test item .

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the saturated solution
The saturated solution (1 mg/L test item was weighed out) was prepared with demineralized water three days prior to the start of the exposure (0 h). The dispersion was stirred for 72 hours (1100 rpm, 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 filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. After the separation treatment the components of the dilution water were added to the saturated solution. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered.

Test concentrations (nominal)
Five dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2.50: 2.56 - 6.40 - 16.0 - 40.0 - 100% of the saturated solution. The dilution levels are based on the results of a preliminary range finding test (non-GLP, Erlenmeyer flasks, with headspace in OECD TG 201 medium)

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

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:
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 concentration pH-value
[% saturated solution] Start; 0 hours End; 72 hours
100 7.74 9.09
40.0 7.73 9.32
16.0 7.80 9.25
6.40 7.86 9.16
2.56 8.06 9.19
Control 8.17 9.16
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 2253449 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: 6132 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:
NOEC
Effect conc.:
100 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate und yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % saturated solution
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 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:
EC-values and statisticall analyses
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were determined from the recorded data without any statistical calculations.

NOEC, LOEC and statistically analyses
The NOEC and LOEC were determined by calculation of significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. 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. The Normality test failed for calculation of growth rate. Therefore, the data were transformed (Y=10Y).

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 concentration

 

[% saturated solution]

Replicate

Cell density [cells/mL]

No.

0 hours

24 hours

48 hours

72 hours

100

1

6839

28927

339712

2287761

2

6839

28389

297449

2252298

3

6839

34182

329694

2450740

Mean

6839

30499

322285

2330266

 40.0

1

6839

33293

353078

2529683

2

6839

35394

402668

2771546

3

6839

32834

390145

2609095

Mean

6839

33840

381964

2636775

 16.0

1

6839

35160

421687

2508733

2

6839

32918

389911

2402812

3

6839

33217

435369

2696933

Mean

6839

33765

415656

2536159

   6.40

1

6839

28117

344827

1819367

2

6839

31455

359914

2545015

3

6839

29185

348385

2197933

Mean

6839

29586

351042

2187438

   2.56

1

6839

34235

355782

2271083

2

6839

29750

344488

2284660

3

6839

27751

341304

2256336

Mean

6839

30579

347191

2270693

Control

1

6839

28881

252752

1931550

2

6839

39935

459901

2485266

3

6839

33302

373923

1288708

4

6839

31657

388705

1934710

5

6839

33226

457349

2978824

6

6839

33846

385276

2901636

Mean

6839

33475

386318

2253449

 

 


 

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

Inhibition of growth rate

Yield

Inhibition of yield

[% saturated solution]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.94

-1

 

2280922

-2

2

 

1.93

-1

 

2245459

0

3

 

1.96

-2

 

2443901

-9

Mean

(-)

1.94

-1

(-)

2323427

-3

 40.0

1

 

1.97

-3

 

2522844

-12

2

 

2.00

-4

 

2764707

-23

3

 

1.98

-3

 

2602256

-16

Mean

(-)

1.99

-3

(-)

2629936

-17

 16.0

1

 

1.97

-3

 

2501894

-11

2

 

1.95

-2

 

2395973

-7

3

 

1.99

-4

 

2690094

-20

Mean

(-)

1.97

-3

(-)

2529320

-13

   6.40

1

 

1.86

3

 

1812528

19

2

 

1.97

-3

 

2538176

-13

3

 

1.92

0

 

2191094

2

Mean

(-)

1.92

0

(-)

2180599

3

   2.56

1

 

1.94

-1

 

2264244

-1

2

 

1.94

-1

 

2277821

-1

3

 

1.93

-1

 

2249497

0

Mean

(-)

1.94

-1

(-)

2263854

-1

Control

1

 

1.88

 

 

1924711

 

2

 

1.97

 

 

2478427

 

3

 

1.75

 

 

1281869

 

4

 

1.88

 

 

1927871

 

5

 

2.03

 

 

2971985

 

6

 

2.02

 

 

2894797

 

Mean

 

1.92

 

 

2246610

 

 


 

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

1.44

2.17

2.03

1.88

0.388

20.6

25.4

2

1.77

2.44

1.69

1.97

0.416

21.2

3

1.58

2.42

1.24

1.75

0.607

34.8

4

1.53

2.51

1.61

1.88

0.544

28.9

5

1.58

2.62

1.87

2.03

0.537

26.5

6

1.60

2.43

2.02

2.02

0.417

20.7

 

 

 

Mean

1.92

 

 

 

 

 

SD ±

0.11

 

 

 

 

CV [%]

5.50

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 % saturated solution, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 100 % saturated solution, respectively. The saturated solution was prepared with a nominal loading of 1 mg/L.
Executive summary:

The toxicity of 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol (batch number:3.April 2010 g) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2017-11-17 to 2017-12-07 at the test facility. 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 6839 cells/mL. A saturated solution with a nominal loading of 1.00 mg test item/L was tested. Five concentrations were tested in a geometrical series with a dilution factor of 2.50 (nominal):2.56 - 6.40 - 16.0 - 40.0 - 100 % saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

 

No analytical monitoring was carried out due to the low solubility of the test item.

 

All effect values were based on the nominal test item of1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol. The results are summarized in the following table:

 

NOEC, LOEC, EC - values of 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol (0-72hours)

                  based on the nominal test item concentration [% saturated solution]

 

 

Nominal test item concentration
[% saturated solution]

Inhibition of Growth Rate

NOEC

  100

LOEC

> 100

ErC10

> 100

ErC20

> 100

ErC50

> 100

 

Inhibition of Yield

NOEC

  100

LOEC

> 100

EyC10

> 100

EyC20

> 100

EyC50

> 100