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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:
2016-11-21 to 2016-11-24
Reliability:
1 (reliable without restriction)

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test item: A130 6-Methoxy-2-methylamino-3-Aminopyridin HCl

Trade name: 3-Amino-2-methylamino-6-methoxypyridine HCl

Batch number: 20151207

CAS number: 83732-72-3

Purity (certified):
93.36/ 93.69 % (Photometric)
99.63% (HPLC area-%)

Sum formula: C7H13Cl2N3O

Appearance: Grey, red, brown powder

Water solubility: >100 g/L

Vapour Pressure: ≤ 1.4*101 Pa (20 °C)

Expiry date : 2017-12-07

Recommended storage : Keep containers tightly closed in a cool and well-ventilated place

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
The test concentrations of A130 6-Methoxy-2-methylamino-3-aminopyridine HCl were analytically verified via LC/MS-MS at the start (0 hours), after 12, 18 and at the end of the exposure (72 hours) with algae.

Test solutions

Vehicle:
no
Details on test solutions:
Stock solution, media preparation
10 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the solution was homogeneous.

Test concentrations (nominal)
5 concentrations were tested in a geometrical series with a dilution factor of 2.5: 20.5 - 51.2 - 128 - 320 - 800 µg/L, corresponding to the time weighted average test item concentration of 3.02 – 3.26 – 3.50 – 4.56 – 5.00 µg/L.
The concentrations are based on the results of two preliminary range finding tests.

Control
Six replicates (without test item) were tested under the same test conditions as the test item replicates. Additionally six replicates were tested in dilution water without ascorbic acid.

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
Remarks on exposure duration:
The method was modified according to OECD 23. The test item solutions are orange. Since this may affect the light intensities for algae cultures the volumes of test media in test vessels were lowered in order to minimize the water layer and path length.
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-values
[µg/L] Start; 0 hours End; 72 hours
800 7.40 7.26
320 7.40 7.41
128 7.43 7.47
51.2 7.41 7.46
20.5 7.57 7.59
Control without ascorbic acid 7.51 7.58
Control 7.41 7.47
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 25 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 6305 cells/mL
- Control end cells density: Mean 2712769 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:

Three-fold concentrated AAP medium according to the guidelines

Composition of Dilution water
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
Ascorbic acid 1
pH 7.5  0.1
The pH of the test medium had to be 7.5  0.1 and was adjusted prior to testing with addition of 1 M NaOH or HCl.



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: 5370 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 per test item concentration was 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:
NOEC
Effect conc.:
128 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
380 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 329 - 669
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
128 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
213 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: CI: 132 - 289
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: No
- 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 2016-10-11 to 2016-10-14 (with headspace). 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 0.436 0.764 ± 0.513
95% confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95% confidence interval 0.179 – 0.322
SD = Standard deviation
Reported statistics and error estimates:
EC-values and statistical analyses
EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.


NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. 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. 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 difference between control and control without ascorbic acid was determined by calculation of statistically significant differences of growth rates and yield. A t-test was used for calculations. When running a t-test, 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.

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.

Any other information on results incl. tables

Cell Densities

Nominal test item concentration

Replicate

Cell density [cells/mL]

[µg/L]

No.

0 hours

24 hours

48 hours

72 hours

800

1

6305

20893

14717

13911

2

6305

29057

25683

25426

3

6305

18483

16262

13550

Mean

6305

22811

18887

17629

320

1

6305

18771

76884

216509

2

6305

25198

106422

389235

3

6305

35191

192204

598179

Mean

6305

26387

125170

401308

128

1

6305

34357

232634

1701426

2

6305

31267

171699

1562844

3

6305

39916

309514

2381200

Mean

6305

35180

237949

1881823

 51.2

1

6305

24643

161350

1347797

2

6305

28699

193842

1843659

3

6305

39180

332136

2420764

Mean

6305

30841

229109

1870740

 20.5

1

6305

19165

220966

2221548

2

6305

32541

219252

1523367

3

6305

22520

203438

2212415

Mean

6305

24742

214552

1985777

Control without ascorbic acid

1

6305

40686

347472

3123741

2

6305

45052

339742

2298564

3

6305

40178

246185

2711603

4

6305

30061

323972

2927088

5

6305

33525

316959

3037480

6

6305

38473

347122

2502321

Mean

6305

37996

320242

2766800

Control

1

6305

34766

372284

2113806

2

6305

45080

412224

3020058

3

6305

46349

441801

3190439

4

6305

31476

301325

1868328

5

6305

41480

418065

2946708

6

6305

40291

405819

3137272

Mean

6305

39907

391920

2712769


Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values (with ascorbic acid) are marked (+), not significant differences are marked (-).

 

Nominal test item concentration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[µg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

800

1

 

0.264

87

 

7606

100

2

 

0.465

77

 

19121

99

3

 

0.255

87

 

7245

100

Mean

(+)

0.328

84

(+)

11324

100

320

1

 

1.18

41

 

210204

92

2

 

1.37

32

 

382930

86

3

 

1.52

25

 

591874

78

Mean

(+)

1.36

33

(+)

395003

85

128

1

 

1.87

7

 

1695121

37

2

 

1.84

9

 

1556539

42

3

 

1.98

2

 

2374895

12

Mean

(-)

1.89

6

(-)

1875518

31

 51.2

1

 

1.79

11

 

1341492

50

2

 

1.89

6

 

1837354

32

3

 

1.98

2

 

2414459

11

Mean

(-)

1.89

6

(-)

1864435

31

 20.5

1

 

1.95

3

 

2215243

18

2

 

1.83

9

 

1517062

44

3

 

1.95

3

 

2206110

18

Mean

(-)

1.91

5

(-)

1979472

27

Control without ascorbic acid

1

 

2.07

 

 

3117436

 

2

 

1.97

 

 

2292259

 

3

 

2.02

 

 

2705298

 

4

 

2.05

 

 

2920783

 

5

 

2.06

 

 

3031175

 

6

 

1.99

 

 

2496016

 

Mean

(-)

2.03

 

(-)

2760495

 

Control

1

 

1.94

 

 

2107501

 

2

 

2.06

 

 

3013753

 

3

 

2.08

 

 

3184134

 

4

 

1.90

 

 

1862023

 

5

 

2.05

 

 

2940403

 

6

 

2.07

 

 

3130967

 

Mean

 

2.01

 

 

2706464

 


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

1.71

2.37

1.74

1.94

0.375

19.4

12.2

2

1.97

2.21

1.99

2.06

0.136

6.59

3

1.99

2.25

1.98

2.08

0.155

7.49

4

1.61

2.26

1.82

1.90

0.332

17.5

5

1.88

2.31

1.95

2.05

0.229

11.2

6

1.85

2.31

2.05

2.07

0.229

11.0

 

 

 

Mean

1.92

 

 

 

 

 

SD ±

0.02

 

 

 

 

CV [%]

0.86

 

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, A130 6-Methoxy-2-methylamino-3-aminopyridine HCl 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 concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 380 (329 – 669) µg/L and 213 (132 – 289) µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 128 µg/L.
The EC50-values based on time weighted average test item concentrations with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 4.73 (4.59 – 4.97) µg/L and 4.02 (3.53 – 4.39) µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 3.50 µg/L.
Since no data on volatility was provided by the sponsor, the loss of test item over time is considered to be an effect of abiotic or biotic degradation. Therefore, toxicity effects are considered to be related to the test item and, most likely, a formed mixture of degradation products.
Executive summary:

The toxicity of A130 6-Methoxy-2-methylamino-3-aminopyridine HCl (Batch no.:20151207) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3 from2016-11-21 to2016-11-24 at the test facility. The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-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 6305 cells/mL. A stock solution of 10.0 mg test item/L was freshly prepared with dilution water. The definitive test was carried out with five concentrations in a geometrical series with a dilution factor of 2.5:20.5 - 51.2 - 128 - 320 - 800 µg/L, corresponding to the time weighted average test item concentration of 3.02 – 3.26 – 3.50 – 4.56 – 5.00 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

 

The test media were clear and light yellow at the start of the exposure. A concentration related color-change was observed throughout the test period from light yellow to yellow. The concentrations of thetest itemA1306-Methoxy-2-methylamino-3-aminopyridine HClin all test concentrations and the control were analytically verified by LC-MS/MS at the start, after 12, 18 and 72 hours (end of exposure). Details of the analytical method are presented in part14. The analytical results are presented inTable 11.
The measured concentrations ofA1306-Methoxy-2-methylamino-3-aminopyridineHClin the fresh media (0 h) were between 97 and 116% of the nominal values. The measured concentrations in the media after 12 hours were between < LOQ and 4% of the nominal values. After 18 hours and 72 hours (end of exposure) all concentrations in the test media were < LOQ.

 

Since no information about volatility was provided by the sponsor, loss of test item is considered to be an abiotic or biotic degradation effect. The observed effects can therefore not be related to the test item A130 6-Methoxy-2-methylamino-3-aminopyridine HCl itself. Concentration control analysis showed complete degradation of the test item after 18 hours at the latest under test conditions, while inhibitory effects are observed over the whole 72 hours. No specific degradation product could be detected in the LC-MS/MS analysis. Therefore, toxicity effects are considered to be related to the test item and, most likely, a formed mixture of degradation products.

 

Based on this, all effect values given are based on the nominal test item concentrations of A130 6-Methoxy-2-methylamino-3-aminopyridine HCl.

 

 NOEC, LOEC, ECx-values and 95% Confidence Intervals of A130 6-Methoxy-2-methylamino-3-aminopyridine HCl (0 - 72 hours)

based on nominal test item concentrations [µg/L]; the test item was found to be completely degraded after 18 hours

 

Growth Rate Inhibition

Nominal test item concentration [µg/L]

NOEC

128

LOEC

320

ErC10

211 (109 – 307)

ErC20

272 (150 – 314)

ErC50

380 (329 – 669)

 

Yield Inhibition

NOEC

128

LOEC

320

EyC10

< 20.5

EyC20

< 20.5

EyC50

213 (132 – 289)