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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:
From 2018-02-05 to 2018-02-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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:
Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)
EC Number:
277-086-3
EC Name:
Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)
Cas Number:
72928-60-0
Molecular formula:
C32H16-τN8Cu(SO3C15H18N3)τ
IUPAC Name:
Bis and tris and tetra [N-(2-methylanilino)-N’-(2-methylanilino) methaniminium][phthalocyaninesulfonato-κ4N29,N30,N31,N32]cuprate(II)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At test facility: 2017-04-19 to 2017-04-27: refrigerator (6 ± 2 °C)/ since 2017-04-27: room temperature, protected from light, in the tightly closed original container; since 2017-11-02 additionally in evacuated desiccator

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
* 0 mg/L (control)
* 0.500 mg/L of test item
* 1.58 mg/L of test item
* 5.00 mg/L of test item
* 15.8 mg/L of test item
* 50 mg/L of test item

- Sampling method: Sampling done at the start of exposure (0 hours) and at the end of exposure (72 hours) with algae.

- Preparation of the samples:
The control and the 2 lowest test item concentrations (0.500 mg/L and 1.58 mg/L) were analyzed undiluted.
The test item concentrations from 5.00 mg/L to 50.0 mg/L were diluted with ultra-pure water prior to analysis.


- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock solution with a nominal test item concentration of 50.0 mg/L was freshly prepared
with dilution water. This dispersion was stirred at 1100 rpm at room temperature for 24 hours.

The four other concentrations0.500 - 1.58 - 5.00 - 15.8 mg/L were prepared by appropriate dilution of the stock solution.

- Control: Six replicates (without test item)

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata

- Source (laboratory, culture collection):
* Origin :Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches Institut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen

*Cultivation at test facility
- Age of inoculum (at test initiation): Four day old

- Method of cultivation:
* Fresh stocks are prepared on Z-Agar.
* Light intensity amounted to 2590 - 5180 lux corresponding to 35 - 70 μE ∙ m-2 ∙ s-1 for 24 hours per day.
* Nutrient medium Z according to LÜTTGE et al. (1994)
* Culture apparatus: Climate room

ACCLIMATION
- Acclimation period: No acclimation period as the preculture was incubated under the same conditions as the test replicates.
- Culturing media and conditions (same as test or not): same as test

- Any deformed or abnormal cells observed: no

Study design

Test type:
static
Water media type:
other: OECD TG 201 medium
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
21 - 24°C, controlled at ± 2°C
pH:
8.1 ± 0.2
Nominal and measured concentrations:
Nominal concentrations : 0.500 – 1.58 – 5.00 – 15.8 – 50.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks 250mL sterile glass Erlenmeyer flasks sealed with cotton wool plugs.

- Type : closed with cotton wool plugs

- Material, size, headspace, fill volume: 250mL sterile glass Erlenmeyer flasks

- Renewal rate of test solution (frequency/flow rate): no renewal

- Initial cells density: 5523cells/ml

- Control end cells density: yes

- No. of vessels per concentration (replicates): 3 replicates

- No. of vessels per control (replicates): 6 replicates


GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium

OTHER TEST CONDITIONS
- Sterile test conditions: no

- Adjustment of pH: yes, for the culture medium

- Photoperiod: 24hours/day light

- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 μE*m-2*s-1. Light homogeneity within ± 15% over incubation area

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

- Determination of cell concentrations:fluorimeter (Microplate Reader Chameleon V (HIDEX) with Software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH)
- Chlorophyll measurement: Chlorophyll a-fluorescence, (with excitation at 436 nm, emission at 685 nm), used to determine cell density.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: spacing factor of √10

- Range finding study : yes

- Test concentrations: 1, 10 and 100 mg of test item /L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 15.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 35.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CI :5.144 - > 50.0
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
ca. 38.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI :5.55 - > 50.0
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 43.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 6.09 - > 50.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 15.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
ca. 28.66 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 956% CI : > 0.500 – 50.0
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 35.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI 5.25 – 50.0
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 0.535 mg/L (CI 95% 0.523-0.547 mg/l) (based on growth rate)
- EyC50: 0.320 mg/L (CI 95% 0.249-0.339 mg/l) (based on yield)
Reported statistics and error estimates:
ErC50 (based on growth rate) is determined via the following the Sigmoidal dose-response (variable slope)
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))


EyC50 (based on yield) is determined via the following the Sigmoidal dose-response (variable slope)
Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))

NOEC/LOEC based growth rate were determined after Wiliams multiple sequential t-test procedure

NOEC/LOEC based on yield were determined after Wiliams multiple sequential t-test procedure

Any other information on results incl. tables

Table 1: Cell Densities

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

50.0

1

5523

6565

4385

2977

2

5523

5859

5827

<LOQcell density

3

5523

5402

5906

3831

Mean

5523

5942

5373

n.a.

15.8

1

5523

22148

229640

1644026

2

5523

23244

226489

1929155

3

5523

25415

273340

1829239

Mean

5523

23602

243156

1800807

5.00

1

5523

27531

269927

2168894

2

5523

26835

318898

1944614

3

5523

31517

320769

2055692

Mean

5523

28628

303198

2056400

1.58

1

5523

17893

165112

1507315

2

5523

33134

417040

2090759

3

5523

23785

378803

1997807

Mean

5523

24937

320318

1865294

0.500

1

5523

18559

173755

1280862

2

5523

17838

174733

1374209

3

5523

15497

157239

960123

Mean

5523

17298

168576

1205065

Control

1

5523

27521

277850

2113231

2

5523

38222

413504

2311038

3

5523

20441

298179

1956961

4

5523

30756

287876

2159164

5

5523

30090

367295

2045863

6

5523

17880

275775

1437379

Mean

5523

27485

320080

2003939

< LOQcell density= below the limit of quantification (2736 cells/mL)          

n.a. = data not determinable
The cell growth in the control increased 362-fold from 0 to 72 hours:
(2003939 cells/mL) x (5523 cells/mL)-1= 362-fold (required: at least 16-fold).

Table 2: Evaluation after 72 hours

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

tiNominal test item concentration

Replicate

Growth Rate

Inhibition of Growth Rate

Yield

Inhibition of Yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

50.0

1

 

-0.21

100

 

-2546

100

2

 

n.a.

100

 

n.a.

100

3

 

-0.12

100

 

-1692

100

Mean

(+)

n.a.

100

(+)

n.a.

100

15.8

1

 

1.90

3

 

1638503

18

2

 

1.95

0

 

1923632

4

3

 

1.93

1

 

1823716

9

Mean

(-)

1.93

2

(-)

1795284

10

5.00

1

 

1.99

-2

 

2163371

-8

2

 

1.96

0

 

1939091

3

3

 

1.97

-1

 

2050169

-3

Mean

(-)

1.97

-1

(-)

2050877

-3

1.58

1

 

1.87

5

 

1501792

25

2

 

1.98

-1

 

2085236

-4

3

 

1.96

0

 

1992284

0

Mean

(-)

1.94

1

(-)

1859771

7

0.500

1

 

1.82

7

 

1275339

36

2

 

1.84

6

 

1368686

32

3

 

1.72

12

 

954600

52

Mean

(-)

1.79

9

(-)

1199542

40

Control

1

 

1.98

 

 

2107708

 

2

 

2.01

 

 

2305515

 

3

 

1.96

 

 

1951438

 

4

 

1.99

 

 

2153641

 

5

 

1.97

 

 

2040340

 

6

 

1.85

 

 

1431856

 

Mean

 

1.96

 

 

1998416

 

n.a. = data not determinable

Table 3:

Section-by-section and average specific growth rates oght 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

1.61

2.31

2.03

1.98

0.355

17.9

24.9

2

1.93

2.38

1.72

2.01

0.337

16.7

3

1.31

2.68

1.88

1.96

0.689

35.2

4

1.72

2.24

2.02

1.99

0.261

13.1

5

1.70

2.50

1.72

1.97

0.460

23.3

6

1.18

2.74

1.65

1.85

0.800

43.2

 

 

 

Mean

1.96

 

 

 

 

 

SD ±

0.06

 

 

 

 

CV [%]

2.84

 

SD = Standard deviation CV = Coefficient of variation

Table 4: Environmental conditions

Nominal test item concentration

pH-values

[mg/L]

Start; 0 hours

End; 72 hours

50.0

7.61

7.65

15.8

7.68

7.98

5.00

7.76

8.23

1.58

7.87

8.44

0.500

7.96

8.59

Control

8.11

8.89

Room temperature [°C]:

min.: 22.0

max.: 22.5

mean value: 22.3

 

Light intensity
[lux]

n = 23

mean value: 5777

range of the measured values: 5077 to 6600 equalling -12.1 to 14.2%

CV [%]: 7.75

CV = Coefficient of variation                             

n = number of measuring points

Table 5 : Measured concentration of test item iun the definitive test with algae

Sampling date

Fresh Media,
0 hours

Old Media,
72 hours

Nominal test item concentration

[mg/L]

TEST ITEM

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

50.0

49.7

99

51.5

103

15.8

14.9

94

15.9

101

    5.00

    4.89

98

    5.08

102

    1.58

    1.51

96

    1.54

 97

      0.500

      0.462

92

      0.453

 91

Control

< LOQ

< LOQ

Meas. conc.  = measured concentration of the test item, mean value of 2 injections, dilution factors taken into account

%                  = percent of the nominal measured concentration of the test item

LOQ             = limit of quantification of the analytical method (0.300 mg/L of the test item)

Table 6 : EC50 values of the reference item

Based on nominal concentrations [mg/l], 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

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, the effect values of te test item on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follows (based on nominal test item concentrations):

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 43.5 (6.09 - > 50.0) mg/L and 36.6 (5.25 – 50.0) mg/L, respectively. The NOEC for growth rate and yield was 15.8 mg/L, respectively.
Executive summary:

The toxicity of the test item 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 2018-02-05 to 2018-02-26, with the definitive exposure phase from 2018-02-20 to 2018-02-23 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The test item is a turquoise blue powder and has a water solubility of 774 mg/L. The study was conducted under static conditions with an initial cell density of 5523 cells/mL. A stock solution and four concentration levels were tested in a geometrical series with a dilution factor of √10: 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L.

Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The measured concentrations in the test solutions at the start of the exposure were in a range of 92 to 99% of the nominal concentrations. At the end of exposure after 72 hours, the measured concentrations were in the range of 91 to 103% of the nominal concentrations.

 

The test item concentrations remained within ± 20% of the nominal concentrations. Therefore, all effect values are given as nominal concentrations.

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