1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

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 2020-09-01 to 2020-09-04 at the test facility

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

EC Number:

216-122-4

EC Name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Cas Number:

1502-47-2

Molecular formula:

C6H6N10

IUPAC Name:

1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Test material form:

solid

Details on test material:

Name of test material (as cited in the report): Exolit 775

Specific details on test material used for the study:

Test item Exolit 775

Batch number Ba-Ha-19-025-1

CAS number 1502-47-2

Purity (certified) 95.75% [m/m]

Chemical name 1,3,4,6,7,9,9b-Heptaazaphenalene-2,5,8-triamine

Molecular formula C6H6N10

Molecular weight 218.18 g/mol

Appearance White solid

Water Solubility 0.93 mg/L

Stability under test
conditions Not specified

Expiry date 2034-09-06

Recommended storage Store container tightly closed in a cool, well-ventilated place.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
Samples were analyzed with an LC-MS/MS method, implemented
under non-GLP and documented finally in the GLP raw data. The method was validated according to Annex I.

Sampling schedule
The saturated solution (with and without algae) and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Sampling and pre-treatment
At the start, samples were taken from one additional replicate of the saturated solution and the control and at the end samples were taken from pooled replicates.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading rate of 20 mg/L of the test item was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and inserted into a glass flask with an appropriate amount of dilution water. The saturated solution was stirred for 24 ± 1 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 of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Preparation of the test item solutions
One saturated solution of 20 mg test item/L was tested. The limit loading rate is based on the results of a preliminary range finding test (non-GLP). For results of the range finding test, see Annex II.

Control The control solution was prepared without test item following the same method as specified for the saturated solution. Six replicates were exposed under the same conditions as the test concentrations.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4
- Strain: CCAP 278/4
- Source (laboratory, culture collection):
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

- 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:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.5 mean value: 22.8

pH:

Environmental Conditions

Geometric mean
measured
test item
concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
1.84 7.89 8.90
Control 7.92 9.01
Room temperature [°C]: min.: 22.0 max.: 23.5 mean value: 22.8

Light intensity
[lux] n = 17 mean value: 6091
range of the measured values: 5422 to 6693 equalling -11.0 to 9.89% CV [%]: 6.28
CV = Coefficient of variation n = number of measuring points

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal test item concentration: One saturated solution of 20 mg test item/L was tested, corresponding to an initially measured concentration of 1.70 mg/L.
Measured test item concentration: At the end of the exposure (72 hours) the measured concentration was in the range of 117% of the initially measured concentration of the test item during the definitive test: 1.99 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 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 5375 cells/mL
- Control end cells density: Mean 1040137 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm)
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, measured: 5422 - 6693 lux with a mean of 6091 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 observed in the preliminary range finding test at the concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Effect concentrations 11

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

89.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

with adjustment of pH

Effect concentrations 12

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 89.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

with adjustment of pH

Effect concentrations 13

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

89.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other:

Remarks:

with adjustment of pH

Effect concentrations 14

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 89.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other:

Remarks:

with adjustment of pH

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 were clear throughout exposure period (possible turbidity related to algae growth not taken into account).
- 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 2020-04-20 to 2020-04-23. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2006, and the limits were set using the threefold standard deviation of these values

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 1.12 0.771 ± 0.563
95% confidence interval 1.08 – 1.15
Yield inhibition
EyC50 0.594 0.420 ± 0.344
95% confidence interval 0.518 – 0.664
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10, EC20 and EC50-values of the growth rate and yield inhibition were estimated empirically based on the results of the single treatment level (Limit test design).

NOEC and LOEC values
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEC/LOEC calculations. When running a t-test, a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. The LEVENE’S test was done for analysis of variance homogeneity. P-values for both Normality and Equal Variance tests are 0.01. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report are 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.
• ToxRat Professional, Version 3.3.0, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

Section-by-section and average specific growth rate are given in the table below. Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the geometric mean measured test item concentration.

Cell Densities

Geometric mean

measured test

item

concentration       Replicate       Cell density [cells/mL]

[mg/L]                     No.       0 hours       24 hours       48 hours       72 hours

1.84                          1        5375       19535       123626       896036

                                 2        5375       21654       119873       894618

                                 3        5375       22428       141678       883916

                                 4        5375       19447       133810       935571

                                 5        5375       21705       129744       936940

                                 6        5375       21155       139362       908888

                           Mean        5375       20987       131349       909328

Control                      1        5375       20693       146487       1105395

                                 2        5375       21126       133136       1081302

                                3        5375       22462       155166       1072114

                                  4        5375       22091       144493       822242

                                 5        5375       23787       141248       1095376

                                  6        5375       23841       137778       1064393

                            Mean        5375       22333       143051       1040137

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       Growth rate inhibition       Yield       Inhibition of yield

[mg/L]               No.        [d-1]        [%]        [cells/mL]       [%]

1.84        1               1.71        3               890661       14

       2                  1.71        3               889243       14

       3               1.70        3               878541       15

       4               1.72        2               930196       10

       5               1.72        2               931565       10

       6               1.71        2               903513       13

       Mean     (+)*  1.71        2        (+)       903953       13

Control        1               1.78                      1100020       

       2               1.77                     1075927       

       3               1.77                      1066739       

       4               1.68                      816867       

       5               1.77                      1090001       

       6               1.76                      1059018       

       Mean               1.75                      1034762       

* = statistically significant but biologically not relevant (inhibition < 5%)

Section-by-Section and Average Specific Growth Rates of the Control Group

(0 - 72 hours)

       Replicate       Specific growth rate [d-1]         Mean SD CV Mean CV

No. section-by-section (0 - 72 hours)        ±        [%]        [%]

                                       0 - 24 hours       24 - 48 hours       48 - 72 hours                            

Control        1        1.35        1.96        2.02        1.78        0.372       20.9        17.3

       2        1.37        1.84        2.10        1.77        0.368       20.8       

       3        1.43       1.93        1.93        1.77        0.290       16.4       

       4        1.41        1.88        1.74        1.68        0.239       14.2       

       5        1.49        1.78        2.05        1.77        0.281       15.8       

       6        1.49        1.75        2.05        1.76        0.278       15.7       

                     Mean        1.75       

                     SD ±        0.04       

                     CV [%]        2.16       

SD = Standard deviation       CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Exolit 775 was found not to inhibit the growth rate of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours but the yield. Effect values were determined as follows (based on geometric mean measured test item concentration): The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 1.84 mg/L. The NOEC-values for inhibition of growth rate and yield after 72 hours were 1.84 mg/L and < 1.84 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 2020-08-31 to 2020-09-04, with the definitive exposure phase from 2020-09-01 to 2020-09-04 at the test facility. The aim of the study was the determination of the effects of growth rate and yield over a period of 72 hours in a limit test.

 

The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A saturated solution with a nominal loading rate of 20 mg/L of the test item was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and inserted into a glass flask with an appropriate amount of dilution water (see Table 2). The saturated solution was stirred for 24 ± 1 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 Tyndall effect was negative. Six replicates were tested for the limit concentration and six for the control. The test media were clear throughout the test period. The environmental conditions were within the acceptable limits.

 

The concentration of the test item Exolit 775 was analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) in the limit concentration and in the control. The initially measured concentration of the test item was 1.70 mg/L. At the end of the exposure (72 hours) the measured concentration was in the range of 117% of the initially measured concentration of the test item during the definitive test.

 

NOEC, LOEC, ECx-values of Exolit 775 (0 - 72 hours)

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

      Growth Rate Inhibition

NOEC          1.84

LOEC       > 1.84

ErC10       > 1.84

ErC20       > 1.84

ErC50       > 1.84

      Yield Inhibition

NOEC       < 1.84

LOEC          1.84

EyC10       < 1.84

EyC20       > 1.84

EyC50       > 1.84