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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-06-08 to 2020-06-09

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 209 (Activated Sludge, Respiration Inhibition 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 No. 1502-47-2
Purity 95.75% [m/m]
Chemical name 1,3,4,6,7,9,9b-Heptaazaphenalene-2,5,8-triamine
Other name Melem
Sum formula C6H6N10
Molar weight 218.18 g/mol
Appearance White solid
Water solubility 0.93 mg/L
TOC 0.33 mg C / mg test item
Expiry date 2034-09-06
Recommended storage Store container tightly closed in a cool, well-ventilated place.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 1000 mg/L were weighed out directly in Erlenmeyer flasks and stirred overnight in 200mL ultrapure water at 400 rpm.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 1000 mg/L
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test
-Reference item: three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8., Triplicates

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Receipt: 2020-06-08
- Pretreatment:The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.93 g/L (corresponding to 1.47 g/L in the test vessels)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Hardness:

Not determined

Test temperature:

20.5 °C

pH:

pH-value of the activated sludge 7.25
pH-value of the synthetic waste water 7.25

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 3
- 500 mL Erlenmeyer flasks, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to keep the sludge flakes in suspension
- Pretreatment: Stirring overnight in 200 mL ultrapure water at 400 rpm

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
-No. of vessels per reference item (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8,at the end of the test)
- Composition of test medium:

Test concentration [mg/L] 1000
Test Item [mg] 500
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250



Dilution Table for the Reference Item
Reference item concentration [mg/L] 58 100 180
Stock solution [µL]* 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
* Stock solution with 300g/L


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.

TEST CONCENTRATIONS

Inhibitions in the Preliminary Tests
A preliminary range finding test (non GLP) was carried out with the concentrations of the test item Exolit 775 of 10 – 100 – 1000 mg/L with one replicate each. The pH was measured in one replicate with 1000 mg/L test item. The flasks were aerated for 3 hours.

In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. One replicate each of 10 – 100 – 1000 mg/L were measured with addition of Allylthiourea (ATU).

Inhibitions in the Preliminary Tests

Nominal Test Item Concentration
[mg/L] pH
(test item in medium) Inhibition of total respiration (without ATU)
[%] Inhibition of heterotrophic respiration (with ATU)
[%] Inhibition of nitrification
[%]
10 - -6 -5 -6
100 - -3 11 -15
1000 7.52 6 0 11


No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.

Based on the results of the preliminary test it was concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in 3 replicates (limit test to determine the NOEC). The concentration was weighed out directly in Erlenmeyer flasks and stirred overnight.

Triplicates of the control without test item were included at the beginning and at the end of the test.

The reference item was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of approx. 1.8.

A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice and adjusted with chlorine free tap water to a dry sludge concentration of 3.0 g/L ± 10 %.

The oxygen uptake rates of all samples were determined following an incubation period of 3 h.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Reported statistics and error estimates:

The NOEC for the oxygen uptake rate was determined with the software ToxRat Professional as specified below.
The NOEC was determined by calculation of statistical significance of the oxygen uptake rate in comparison to the control using statistical standard procedure as normality test (Shapiro-Wilk), variance homogeneity test (Levene’s test) and a two-sample t-test.
The P-value for all tests was 0.01. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05

The EC-value of the reference item was calculated by linear regression. Calculation of the confidence intervals for the EC 50- value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

Any other information on results incl. tables

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Test Item Concentration

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1000	1	7.52	35.1	-2	-1
	2	6.39	35.5	-3
	3	6.94	34.1	1

*) determined at start of the measurement

   Repl. = Replicate

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Reference Item Concentrations

Reference Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
58	1	6.92	29.1	15	23
	2	7.67	25.1	27
	3	7.49	25.0	27
100	1	7.46	17.3	50	51
	2	5.06	17.7	49
	3	6.96	16.2	53
180	1	8.30	8.2	76	77
	2	7.02	7.9	77
	3	8.06	7.4	78

                                            

*) determined at start of the measurement

   Repl. = Replicate

Specific Respiration Rates of the Control Replicates

              

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g DW×h]
Control	1	7.23	36.0	24.5
	2	6.69	33.6	22.9
	3	6.71	33.9	23.1
	4	7.88	35.0	23.8
	5	7.97	34.7	23.6
	6	7.36	32.9	22.4
	Mean value		34.4	23.4
	Standard deviation		1.01
	CV [%]		2.94

         *) determined at start of the measurement

           Repl. = Replicate

           CV = Coefficient of variation

                                             

    

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item Exolit 775 is ≥ 1000 mg/L.
Exolit 775 had no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item from 2020-06-08 to 2020-06-09, with the definitive exposure phase on 2020-06-09 at the test facility. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit test item concentration 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates was -1%.

    

The NOEC of the test item in the respiration inhibition test with activated sludge with Exolit 775 is ≥ 1000 mg/L.