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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2020-08-24 to 2020-08-27 with the definitive exposure phase from 2020-08-25 to 2020-08-27

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

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
The test item concentration was analyzed with LC-MS/MS in the limit concentration and in the control. Samples of the fresh media were taken and analyzed at the start of exposure and at the renewal of the test solutions (0 and 24 hours) as well as samples of the 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours).

Sampling for the analytical monitoring
At the start of the exposure and at the renewal (0 and 24 hours), samples of the fresh media were taken after preparation of the limit concentration and analyzed. At the renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
A saturated solution with a nominal loading rate of 20 mg test item/L was prepared 24 ± 1 hour prior to the start of the exposure and the renewal. An appropriate amount of the test item was weighed out. The test item was inserted into a glass bottle 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. After the stirring period 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 were 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. No presence of undissolved test item during the test was observed. The Tyndall effect was negative.

Limit concentration
A saturated solution of 20 mg test item/L was tested in a limit test.
The limit concentration was selected based on the results of a preliminary range finding test (non-GLP).

Control Dilution water without test item incubated under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system:
Daphnia magna STRAUS, obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), is used. The composition of the culture medium is presented in the table below.

Feeding of the culture stocks
The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility
 

Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)

Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2 to 0.8

Study design

Test type:

semi-static

Water media type:

freshwater

Remarks:

Elendt M4, according to OECD 202, Annex 3 (2004)

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

see any other information on materials and methods incl. tables

Test temperature:

see any other information on materials and methods incl. tables

pH:

see any other information on materials and methods incl. tables

Dissolved oxygen:

see any other information on materials and methods incl. tables

Salinity:

Not applicable, freshwater

Conductivity:

see any other information on materials and methods incl. tables

Nominal and measured concentrations:

A saturated solution of 20 mg test item/L was tested in a limit test, corresponding to the geometric mean measured test item concentration 1.18 mg/L.

Details on test conditions:

Test method
The study was performed under semi-static conditions with a renewal of the test solutions after 24 hours.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL

Dilution water
Same composition as the culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.
 
Acclimatization
Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

> 1.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 1.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 1.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 1.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The limit concentration and the control were colorless and clear throughout the exposure period.

Results with reference substance (positive control):

Test of the Reference Item

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2020-08-04 to 2020-08-05. For results of the most recent of the monthly performed reference tests, see the table below.

EC50-Value (with 95% confidence limits) of the Reference Item Potassium dichromate
based on nominal concentrations [mg/L], (0 - 24 hours)
Current Study Valid Range
EC50 2.03 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
95% confidence limits 1.00- 4.00 mg/L

Reported statistics and error estimates:

Methods of evaluation

The effects of the limit concentration were empirically derived from the observed immobilization rates. All effect levels (EC10 / 50 / 100) were based on the analytically verified geometric mean measured concentration of the test item Exolit 775.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Rates after 24 and 48 hours of Exposure

     (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal

test item

concentration

[mg/L]       IMMOBILIZATION [%]

      24 hours       48 hours

      Replicates       Replicates

      1       2       3       4       MV       1       2       3       4       MV

20.0       0       0       0       0       0       0       0       0       0       0

Control       0       0       0       0       0       0       0       0       0       0

Table 4:       Absolute Numbers of immobile Daphnids after 24 and 48 h

      (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal

test item

concentration

[mg/L]       NUMBER OF IMMOBILE DAPHNIDS / TOTAL NUMBER OF DAPHNIDS

      24 hours       48 hours

      Replicates       Replicates

      1       2       3       4       Sum       1       2       3       4       Sum

20.0       0 / 5       0 / 5       0 / 5       0 / 5       0 / 20       0 / 5       0 / 5       0 / 5       0 / 5       0 / 20

Control       0 / 5       0 / 5       0 / 5       0 / 5       0 / 20       0 / 5       0 / 5       0 / 5       0 / 5       0 / 20

Preliminary Range Finding Test (non-GLP)

A non-GLP preliminary range finding test was conducted at the test facility under semi-static conditions over a period of 48 hours with three nominal test item concentrations of 1, 10 and 100% of the saturated solution prepared with dilution water.

A saturated solution with a nominal loading rate of 100 mg/L. The saturated solution was prepared as described in section 4.2. Out of this saturated solution two dilution levels were prepared with the concentrations 1.00 and 10.0% of the saturated solution. The saturated test item solution showed no Tyndall effect. The saturated solution and the dilution levels were visually clear and colorless throughout the exposure period.

In the range finding test, two replicates per concentration level and control, each with five daphnids, were tested. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). .

Immobilization Rates in the non-GLP Preliminary Range Finding Test

      (n = 10, divided into 2 replicates with 5 daphnids each)

Saturated solution

[%]       IMMOBILIZATION [%]

      24 hours       48 hours

      Replicates       Replicates

      1       2       MV       1       2       MV

100       20       0       10       20       0       10

10       0       0       0       0       0       0

1       0       0       0       0       0       0

Control       0       0       0       20*       0       0

*       = replicate 1 was excluded from evaluation, due to accidential mortality of the parental daphnid

Measured Exposure Concentrations during the non-GLP Preliminary Range Finding Test

Determination of the test item via LC-MS/MS

Sampling date       Fresh media,

0 hours       Old media,

24 hours       Fresh media,

24 hours       Old media,

48 hours

Saturated solution

[%]       Exolit 775

      Meas. conc.

[mg/L]       %       Meas. conc.

[mg/L]       %       Meas. conc.

[mg/L]       %       Meas. conc.

[mg/L]       %

100       1.12       1       1.38       1       1.54       2       1.31       1

 10.0       0.141       1       0.237       2       0.142       1       0.133       1

   1.00       0.0482       5       < LCL       < LCL       0.0478       5

Control       < LCL       < LCL       < LCL       < LCL

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

%       = percent of the nominal concentration of the test item

LCL       = lowest calibration level (0.20 µg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the nominal limit concentration 20.0 mg test item/L, corresponding to the geometric mean measured test item concentration 1.18 mg/L, no effects on Daphnia magna were observed.

Executive summary:

 

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item  were determined at the test facility according to OECD 202 (2004) from 2020-08-24 to 2020-08-27 with the definitive exposure phase from 2020-08-25 to 2020-08-27.

The study was conducted under semi-static conditions over a period of 48 hours with a saturated solution (nominal concentration 20.0 mg/L) of the test item Exolit 775. The saturated solution was prepared as specified in section 4.2. The limit concentration was colorless and clear throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to the limit concentration and the control.

The limit concentration and the control were analytically verified LC-MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the 24 hours old media at the renewal and at the end of the test (24 and 48 hours) in the limit concentration and in the control.

 

The validity criteria of the test guidelines were fulfilled.

 

In the nominal limit concentration 20.0 mg test item/L, corresponding to the geometric mean measured test item concentration 1.18 mg/L, no effects on Daphnia magna were observed. All effect concentrations given in the table below are based on the geometric mean measured concentrations of the test item Exolit 775.

 

EC10-, EC50-, EC100-Values

(based on the geometric mean measured concentration of the test item)

 

Effect Test duration

concentrations [hours]        Toxicity endpoint based on

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

EC10 / 50 / 100       24        > 1.18

       48        > 1.18