Bisbenzimidazo[2,1-b:1',2'-j]benzo[lmn][3,8]phenanthroline-6,9-dione

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD 209 and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:1000 mg/L were weighed out directly in Erlenmeyer flasks

- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 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

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)
- 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: 2.87 g/L, corresponding to 1.44 g/L suspended solids in the test vessels)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

21.6 °C

pH:

pH-value of the activated sludge 7.58
pH-value of the synthetic waste water 7.41

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.


- 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.)
- Composition of test medium:

Table for the Test Item compositions
Test item concentration [mg/L]
1000
Inoculum [mL] 250
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL



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 immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
Inhibition of Respiration (Preliminary Tests)

Inhibitions in the Preliminary Tests

Nominal Test pH pH pH Inhibition
Item (test item in (adjusted to) (test item [%]
Concentration demin. water) in medium)
[mg/L]

1st preliminary test - with pH adjustment, direct aeration
10 6.20 - 7.31 -3
6.15 - 7.38 -8
100 6.69 - 7.32 -6
6.69 - 7.33 -8
1000 9.16 7.72* 7.35 -8
9.21 - 7.37 -4
1000 + ATU** 9.23 7.76* 7.53 -
9.36 - 7.56 -

*) adjusted with 0.05 mol/L HCl
**) ATU = N-Allylthiourea (final concentration 11.6 mg/L)

Inhibitions in the Preliminary Test (with / without ATU)

Nominal Test Inhibition of Inhibition of Inhibition of nitrification
Item total respiration heterotrophic respiration
Concentration
[mg/L] [%] [%] [%]
1000 + ATU* -8 -17 2
-4 -10 2

*) ATU = N-Allylthiourea (final concentration 11.6 mg/L)

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item solution was orange-coloured. The test item was not dissolved and settled at the bottom of the Erlenmeyer flasks.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 91.0 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The EC-value of the reference item was calculated by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC50-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.

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 to the oxygen uptake rates of the control

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

Test and Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	24.8	-	-
	2	24.4
	3	23.2
	4	18.0
	5	22.4
	6	23.2
Test item 1000	1	24.4	-7	-7
	2	24.4	-7
	3	24.0	-6
Reference item 58	1	16.4	28	26
	2	17.6	22
	3	16.4	28
100	1	10.4	54	54
	2	10.4	54
	3	10.4	54
180	1	3.2	86	88
	2	2.8	88
	3	2.4	89

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 10.8%

Suspended solids in the test vessel: 1.44 g/L

Specific Respiration Rates of the Control Replicates

		Repl.	Specific Respiration Rate Rs [mg O2/g×h]
	Control	1	17
		2	17
		3	16
		4	13
		5	16
		6	16
Mean value			16

Repl. = Replicate

Suspended solids in the test vessel: 1.44 g/L

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item is 1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit 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. 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 -7 %.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 91.0 mg/L.

 

The validity criteria of the test guideline were fulfilled.

NOEC of the test item

		Test item Concentration [mg/L]
NOEC*		≥ 1000

                        *) No statistically significant effect (P < 0.05), ANOVA

The NOEC of the test item is ≥1000 mg/L.

The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

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

Validity criteria fulfilled:

yes

Conclusions:

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

Description of key information

Read-across information: The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.