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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2020-10-04 to 2020-10-09 with the definitive exposure period from 2020-10-05 to 2020-10-09 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

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 samples were analyzed with a LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data. The method will be validated.
The analytical method and the results of the method validation were added to the final report.

Sampling schedule
The limit loading and the control were analytically verified in the fresh media at the start of exposure and at one renewal interval (0 and 72 hours) and in the corresponding 24-hours aged media after 24 and 96 hours.

Test solutions

Vehicle:

no

Details on test solutions:

Limit concentration
A saturated solution prepared with 20 mg test item/L was tested as a threshold concentration in a limit test. The selection of the test concentration was based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia acute immobilization test (EC50 (0-48 h) = >saturated solution (nominal concentration of 20 mg/L) and of an alga toxicity test (ErC50 (0-72 h) = >saturated solution (nominal concentration of 20 mg/L).

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. 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 was continued. The next 25 mL was 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.

Control
7 fish in dilution water (without test item but from the same source and age as used for the test solution) were tested under the same test conditions as the test replicate.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at NOACK LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study end (length definition, mean, range and SD): Average body length: 1.77 cm
- Weight at study end (mean and range, SD): Average body weight: 0.053 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (540 – 1000 Lux, photoperiod of 12 to 16 h light / 12 to 8 h dark). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure were used in the test. No mortality was observed during this time. No disease treatments were administered throughout holding and testing.
- Feeding during test: No feeding during test


ACCLIMATION
- Acclimation period: Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure were used in the test.

- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

Study Days TOC [mg/L] Total Hardness
[mg CaCO3/L]
0 to 1 < 2 66
1 to 2 < 2 68
2 to 3 < 2 64
3 to 4 < 2 70

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

The measured concentrations of the test item were 2.34 and 2.43 mg/L in the fresh media at start of the exposure intervals and 117% and 101% of the initially measured concentrations at the end of the exposure intervals.


Measured Concentrations of the Test Item during the Definitive Test – Exolit 775
Sampling date Fresh media, Old media
0 hours 24 hours
Nominal Exolit 775
Test Item Meas. conc. Meas. conc. %
Concentration [mg/L] [mg/L]
[mg/L]
20.0 2.34 2.72 117
Control < LOQ < LOQ

Sampling date Fresh media, Old media,
72 hours 96 hours Geometric mean Test item concentration
Nominal Exolit 775
Test Item
Concentration Meas. conc. Meas. conc.
[mg/L] [mg/L] [mg/L] % [mg/L]
20.0 2.43 2.44 101 2.48
Control < LOQ < LOQ -
Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percentage of the initially measured concentration
LOQ = limit of quantification of the analytical method (0.0200 mg/L of the test item)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable):
- Material, size, headspace, fill volume:
Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with glass plates.
Test volume: About 2.5 L per vessel

- Aeration: No aeration was provided
- Renewal rate of test solution (frequency/flow rate): A semi-static test with daily renewal of the test media was performed.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates
- Reference item: No reference item is recommended for this test according to the guideline.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.148 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerate for at least 24 h to remove chlorine.

Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Acidity: 0.2 mmol/L (recent measurement 2020-07-16)
Alkalinity: 0.6 mmol/L (recent measurement 2020-07-16)
Conductivity: 158 µS/cm (recent measurement 2020-07-16)


- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-value, temperature and oxygen saturation were
measured in all test vessels at the start and the end of exposure as well as daily and after renewal of the test media from fresh and old test media.
Chlorine and nitrate were measured from each batch of dilution water used in the test.
Total hardness and TOC were determined at the start of the exposure in the dilution water. During the test, the water temperature was recorded continuously (once per hour) with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure. The solution appearance and behavior was observed daily before and after renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 16 h light / 8 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 540 - 1000 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 2 ± 0.5 h, 5 ± 1 h and 24 ± 2 h after the start of the exposure. On days 1-4 of the test, all vessels with living fish were inspected twice per day.


TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

2.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: See tables below
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: Not observed
- Any observations (e.g. precipitation): The test solution was visibly clear throughout the period of exposure.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Evaluation The LC-values after 96 h were empirically derived from the raw data of the limit loading.

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:

The following software was used for calculations:
Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Sublethal observations / clinical signs:

Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels

(n = 7, number of fish)

Nominal Test Item Loading Rate

[mg/L]       Effect *

      Number of fish affected at observation time [hours]

             day 0       day 1       day 2       day 3       day 4

             2       5       22       28       46       52       70       77       93       96

20.0       (1)       7       7       7       7       7       7       7       7       7       7

Control       (1)       7       7       7       7       7       7       7       7       7       7

      *)  The numbers in brackets correspond to the following observations:  

      (1)       = Normal behavior       

Cumulative Mortality [%] in the Test Vessels

Nominal Test Item Loading Rate

[mg/L]       Cumulative mortality [%] at observation time [hours]

      day 0       day 1       day 2       day 3       day 4

      2       5       22       28       46       52       70       77       93       96

20.0       0       0       0       0       0       0       0       0       0       0

Control       0       0       0       0       0       0       0       0       0       0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect level is given based on the geometric mean measured test item concentration of Exolit 775. The threshold concentration of the test item at the nominal loading rate 20.0 mg/L corresponding to the geometric mean measured concentration of 2.48 mg/L was found to have neither lethal nor non lethal effects on zebrafish.
In conclusion, the LC0 corresponds to the geometric mean measured concentration of ≥ 2.48 mg test item/L.

Executive summary:

 

The acute toxicity of the test item  to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019) from 2020-10-04 to 2020-10-09 with the definitive exposure period from 2020-10-05 to 2020-10-09 at the test facility.

 

The test item is a white solid. A saturated solution prepared with 20 mg test item/L was tested as a threshold concentration in a limit test. The selection of the test concentration was based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia acute immobilization test (EC50 (0-48 h) = >saturated solution (nominal concentration of 20 mg/L) and of an alga toxicity test (ErC50 (0-72 h) = >saturated solution (nominal concentration of 20 mg/L).

 

The study was performed over a period of 96 hours under semi-static conditions with daily renewal of the test solutions. Seven test organisms were exposed to the test item concentration and the control, respectively. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

The test solutions were visually clear throughout the study.

 

The loading rate of the test item  was analytically verified via LC-MS/MS in fresh media at the start of the exposure and at one renewal interval (0 hours and 72 hours) and in the corresponding 24-hours aged media after 24 hours and 96 hours in the limit test item loading and the control.

The measured concentrations of the test item were in the range of 2.34 to 2.43 mg/L of the nominal concentration in the fresh media at start of the exposure intervals. At the end of the exposure intervals the test item concentration were in the range of 117% to 101% of the initially measured test item concentration.

All effect values given were based on the geometric mean measured concentration of the test item.

 

 

All validity criteria of the test guideline were met. The threshold concentration of the test item at the nominal loading rate of 20.0 mg/L corresponding to the geometric mean measured concentration of 2.48 mg/L was found to have neither lethal nor non lethal effects on zebrafish.

 

 

 

LC-Values (96 hours) of the Test item

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

 

LC0 =

Highest test item concentration with 0% mortality after 96 h       2.48