[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-31 to 2017-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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

Constituent 1

Reference substance name:

[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

EC Number:

255-965-2

EC Name:

[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

Cas Number:

42844-93-9

Molecular formula:

C29H18N4NiO3

IUPAC Name:

Nickel, [1,3-dihydro-5,6-bis[[[2-(hydroxy-kO)-1-naphthalenyl]methylene]amino-kN]-2H-benzimidazol-2-onato(2-)]-, (SP-4-2)-

Test material form:

solid: nanoform

Sampling and analysis

Analytical monitoring:

no

Test solutions

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 loadings: 1000 mg/L, 3 replicates
- 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: 2017-05-31
- 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: 2.71 g/L, corresponding to 1.36 g/L in the test vessel

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Hardness:

Not determined

Test temperature:

20.8 °C

pH:

pH-value of the activated sludge 7.45
pH-value of the synthetic waste water 7.34

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 maintain the sludge flakes in suspension.

- Pretreatment: stirring overnight

- 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 Item 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 test (non GLP) was carried out with the following test item concentrations of PV-Echtorange 6RL:
10 – 100 - 1000 mg/L with two replicates each. The pH was not adjusted.

In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. Two replicates 1000 mg/L were measured with addition of Allylthiourea ATU.

No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed, even the inhibitions of the heterotrophic respiration or nitrification show no appreciable value.


Inhibitions in the Preliminary Tests

Nominal Test Item Concentration
[mg/L] pH

(test item in medium) Inhibition


[%]
1st preliminary test - without pH adjustment, direct aeration
10 - 1
- -3
100 - 0
- 3
1000 7.44 -4
- 5

Inhibitions in the Preliminary Test (with/ without ATU)

Nominal Test Item Concentration
[mg/L] Inhibition of total respiration
[%] Inhibition of heterotrophic respiration
[%] Inhibition of nitrification

[%]
1000 -4 -16 3
5 -13 15


Based on the results of the preliminary test it could 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 the Erlenmeyer flasks.

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

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 if necessary to a dry sludge concentration of 3.0 g/L ± 10 %.

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

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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 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.

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 to the oxygen uptake rates of the control.

Oxygen Uptake Rates and 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×h]
Control	1	7.88	32.8	24.2
	2	7.93	33.4	24.6
	3	7.24	32.6	24.0
	4	8.06	34.4	25.4
	5	8.67	33.8	24.9
	6	8.41	32.8	24.2
	Mean value		33.3	24.6
	Standard deviation		0.640
	CV [%]		1.92

                            *) determined at start of the measurement

                            Repl. = Replicate

                            CV = Coefficient of variation

Oxygen Uptake Rates and Inhibition of the Respiration of the Test Item Replicates

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1000	1	8.11	35.8	-8	-6
	2	6.74	35.4	-6
	3	8.01	35.1	-5

*) determined at start of the measurement

Repl. = Replicate

 

Oxygen Uptake Rates and Inhibition of the Respiration of the Reference Item Replicates

Reference Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
58	1	7.20	24.6	26	28
	2	7.80	24.1	28
	3	7.13	23.3	30
100	1	8.29	15.9	52	52
	2	7.56	16.8	50
	3	6.94	15.1	55
180	1	9.17	6.0	82	82
	2	9.31	6.3	81
	3	8.91	5.7	83

*) determined at start of the measurement

Repl. = Replicate

Applicant's summary and conclusion

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 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 (batch number: DEB2 188394) from 2017 -05 -31 to 2017 -06 -01, with the definitive exposure phase on 2017 -06 -01 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 -6% .

 

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 93.1 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 inhibiting effect (P = 0.002), 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.