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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:
2016-10-04 to 2016-10-07
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
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-aminopyridin-3-ol
EC Number:
240-886-8
EC Name:
2-aminopyridin-3-ol
Cas Number:
16867-03-1
Molecular formula:
C5H6N2O
IUPAC Name:
2-aminopyridin-3-ol
Details on test material:
Name of test material (as cited in the report): A132 2-Amino-3-hydroxypyridine

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The test concentrations of A132 2-Amino-3-hydroxypyridine were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Test solutions

Vehicle:
no
Details on test solutions:
Stock solution, media preparation
20.0 mg test item/L, freshly prepared with dilution water. The dispersion was shaken manually until the solution was clear dissolved.

Test concentrations (nominal)
5 concentrations were tested in a geometrical series with a dilution factor of 2: 1.25 - 2.50 - 5.00 - 10.0 - 20.0 mg/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).

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

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.: 21.0 max.: 23.0 mean value: 22.0
pH:
Geometric mean measured test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
2.44 8.04 8.21
1.69 8.05 8.31
1.18 7.94 8.54
0.829 8.01 8.85
0.585 8.03 9.15
Control 8.21 9.31
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Nominal: 1.25 - 2.50 - 5.00 - 10.0 - 20.0 mg/L
geometric mean measured concentrations: 0.585 - 0.829 - 1.18 - 1.69 - 2.44 mg/L
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 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 175431 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 0.064
Na2EDTA 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: 5348 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.:
0.585 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.829 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.979 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 0.868 - 1.08
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.08 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 1.01 - 1.13
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 1.20 - 1.31
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.585 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.829 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.585 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.67 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: CI: < 0.585 - 0.769
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.884 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: CI: 0.819 - 0.959
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 item concentrations were clear during the exposure. The algae in the saturated solution were agglutinated after 72 hours. No agglutination of the algae was observed in the control replicates.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA, 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 2016-04-26 to 2016-04-29 (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 0.460 0.780 ± 0.473
95 % confidence interval 0.443 – 0.481
Yield inhibition
EyC50 0.317 0.411 ± 0.276
95 % confidence interval 0.208 – 0.347
SD = Standard deviation
Reported statistics and error estimates:
ECx-values and statistical Analyses
ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statisticalanalyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:
Monotonicity was done by trend analysis by contrasts (significance level 0.05) for growth rate.
Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01 for growth rate and yield.
Levene’s test on variance homogeneity was done with a significance level 0.01 for growth rate and yield.
Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm was calculated with a significance level 0.05 for yield.
William’s test on significant differences was done with a significance level 0.05 for yield and growth rate.

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 Version 3.2.1, ToxRat Solutions GmbH

Any other information on results incl. tables

 

 Cell Densities

Geometric mean measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

2.44

1

6305

<LOQ

2860

<LOQ

2

6305

2790

2894

2798

3

6305

<LOQ

<LOQ

2798

Mean

6305

n.a.

n.a.

n.a.

1.69

1

6305

4205

5056

6612

2

6305

3537

5067

5806

3

6305

3418

4306

5727

Mean

6305

3720

4810

6048

1.18

1

6305

12803

51457

262924

2

6305

10004

37886

180361

3

6305

10621

44753

175197

Mean

6305

11143

44699

206161

0.829

1

6305

28025

205792

996707

2

6305

21869

210922

1011394

3

6305

18920

132109

1143521

Mean

6305

22938

182941

1050541

0.585

1

6305

33771

281998

1748066

2

6305

28942

305784

1634799

3

6305

25438

182436

1077003

Mean

6305

29384

256739

1486623

Control

1

6305

25618

297113

1641968

2

6305

36643

349518

2245177

3

6305

37280

378717

2186176

4

6305

25643

205287

1280431

5

6305

35817

349028

1425694

6

6305

29598

279669

1746440

Mean

6305

31767

309889

1754314

 

< LOQ    = The cell density was below the limit of quantification (2736 cells/mL). For the evaluation the
  ½Limit of quantification (1368 cells/mL) was applied.

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 (-).

 

Geometric mean measured test item concentration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

2.44

1

 

n.a.

100

 

n.a.

100

2

 

n.a.

100

 

n.a.

100

3

 

n.a.

100

 

n.a.

100

Mean

(+)

n.a.

100

(+)

n.a.

100

1.69

1

 

0.016

99

 

307

100

2

 

-0.027

100

 

-499

100

3

 

-0.032

100

 

-578

100

Mean

(+)

-0.0150

100

(+)

-257

100

1.18

1

 

1.24

33

 

256619

85

2

 

1.12

40

 

174056

90

3

 

1.11

41

 

168892

90

Mean

(+)

1.16

38

(+)

199856

89

0.829

1

 

1.69

10

 

990402

43

2

 

1.69

9

 

1005089

43

3

 

1.73

7

 

1137216

35

Mean

(+)

1.70

9

(+)

1044236

40

0.585

1

 

1.87

0

 

1741761

0

2

 

1.85

1

 

1628494

7

3

 

1.71

8

 

1070698

39

Mean

(-)

1.81

3

(-)

1480318

15

Control

1

 

1.85

 

 

1635663

 

2

 

1.96

 

 

2238872

 

3

 

1.95

 

 

2179871

 

4

 

1.77

 

 

1274126

 

5

 

1.81

 

 

1419389

 

6

 

1.87

 

 

1740135

 

Mean

 

1.87

 

 

1748009

 

 

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

1.40

2.45

1.71

1.85

0.539

29.1

20.2

2

1.76

2.26

1.86

1.96

0.262

13.4

3

1.78

2.32

1.75

1.95

0.320

16.4

4

1.40

2.08

1.83

1.77

0.342

19.3

5

1.74

2.28

1.41

1.81

0.439

24.3

6

1.55

2.25

1.83

1.87

0.352

18.8

 

 

 

Mean

1.87

 

 

 

 

 

SD ±

0.08

 

 

 

 

CV [%]

4.01

 

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, A132 2-Amino-3-hydroxypyridine was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 1.23 (1.20 – 1.31) mg/L and 0.884 (0.819 – 0.959) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.585 mg/L, respectively.
Executive summary:

The toxicity of A132 2 -Amino-3 -hydroxypyridine (Batch no.:20150502) 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 from 2016-10-04 to 2016-10-07 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 20.0 mg test item/L was freshly prepared with dilution water and agitated until the solution was visually clear. The definitive test was carried with five concentrations in a geometrical series with a dilution factor of 2.5: 1.25 - 2.50 - 5.00 - 10.0 - 20.0 mg/L, corresponding to the geometric mean measured concentrations: 0.585 - 0.829 - 1.18 - 1.69 - 2.44 mg/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 colorless at the start of the exposure. A concentration related color-change was observed throughout the test period from colorless to clear yellow-brown. The concentrations of the test item A132 2-Amino-3-hydroxypyridine in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of exposure.
The measured concentrations of A132 2-Amino-3-hydroxypyridine in the fresh media (0 h) were between 91 and 99% of the nominal values. The measured concentrations in the old media (72 h) were < LOQ.

All effect values given are based on the geometric mean measured test item concentrations of A132 2-Amino-3-hydroxypyridine.

 

NOEC, LOEC, ECx-values and 95 % Confidence Intervals ofA1322-Amino-3-hydroxypyridine(0 - 72 hours)

                  based on geometric mean measured test item concentrations [mg/L]

 

Growth Rate Inhibition
Geometric mean measured test item concentration [mg/L]

NOEC

0.585

LOEC

0.829

ErC10

0.979 (0.868 - 1.08)

ErC20

1.08 (1.01 - 1.13)

ErC50

1.23 (1.20 - 1.31)

 

Yield Inhibition

Geometric mean measured test item concentration [mg/L]

NOEC

0.585

LOEC

0.829

EyC10

< 0.585

EyC20

0.670 (< 0.585 - 0.769)

EyC50

0.884 (0.819 - 0.959)

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