5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-20 - 2017-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

For the first study:
- Method: 1 - 5.6 - 32 - 180 - 1000 mg a.i./L were pipetted from a stock solution in Erlenmeyer flasks.
- Stock solution: 5 g a.i./L in demineralized water
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 1 - 5.6 - 32 - 180 - 1000 mg a.i./L, 5 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

For the second study:
- Method: 0.18 - 1 - 5.6 - 32 - 180 - 1000 mg a.i./L were pipetted from a stock solution in Erlenmeyer flasks.
- Stock solution: 5 g a.i./L in demineralized water
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 0.18 - 1 - 5.6 - 32 - 180 - 1000 mg a.i./L, 5 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

For the third study:
- Method: 0.025 - 0.2 - 1.6 - 13 - 100 mg a.i./L were pipetted from a stock solution in Erlenmeyer flasks.
- Stock solution: 5 g a.i./L in demineralized water
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 0.025 - 0.2 - 1.6 - 13 - 100 mg a.i./L, 5 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 (species):

activated sludge

Details on inoculum:

Details on inoculum for the first study:
- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Receipt: 2017-10-19
- 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: 3.18 g/L, corresponding to 1.59 g/L in the test vessel

Details on inoculum for the second study:
- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Receipt: 2017-10-26
- 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: 3.11 g/L, corresponding to 1.56 g/L in the test vessel

Details on inoculum for the third study:
- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Receipt: 2017-12-12
- 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: 2.73 g/L, corresponding to 1.37 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

In the first study: 20.8 °C
In the second study: 19.9 °C
In the third study: 19.5 °C

pH:

pH-value of the activated sludge in the first study 7.68
pH-value of the synthetic waste water in the first study 7.36

pH-value of the activated sludge in the second study 7.61
pH-value of the synthetic waste water in the second study 7.36

pH-value of the activated sludge in the third study 7.54
pH-value of the synthetic waste water in the third study 7.23

Salinity:

N/A

Details on test conditions:

TEST SYSTEM
- Test vessel: 5
- 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: None

- No. of vessels per concentration (replicates): 5 (6 in the second study)
- 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 in the first study:

Test item concentration [mg a.i./L]* 1 5.6 32 180 1000
Test item [ml] 0.1 0.56 3.2 18 100
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
*)Stock solution 5 g a.i./L corresponding 5.7736 g test item/L


- Composition of test medium in the second study:

Test item concentration [mg a.i./L]* 0.18 1 5.6 32 180 1000
Test item [ml] 0.018 0.1 0.56 3.2 18 100
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
*)Stock solution 5 g a.i./L corresponding 5.7736 g test item/L

- Composition of test medium in the thrid study:

Test item concentration [mg a.i./L]* 0.025 0.2 1.6 13 100
Test item [ml] 0.0025 0.02 0.16 1.3 10
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
*)Stock solution 5 g a.i./L corresponding 5.7736 g test item/L


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

A preliminary test (non-GLP) was carried out with the concentrations of the test item Basic Blue 159 Trichlorozinkate of 1 - 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 of 1 - 10 – 100 - 1000 mg/L was measured with the addition of Allylthiourea (ATU).


Inhibitions in the Preliminary Test (with / without ATU)
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
[%]
1 - -4 2 -9
10 - 44 46 41
100 - 84 77 91
1000 6.22 97 97 98

No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed, since the inhibition of heterotrophic respiration as well as the inhibition of nitrification are identical.



Preparation of the Test Vessels in the first study
According to the results of the range finding test, the first study was carried out with 5 concentrations within the range 1 - 1000 mg a.i./L test item in a geometrical series and with a dilution factor of 5.6. No pH adjustment was made.
All concentrations were pipetted from a stock solution in the Erlenmeyer flasks.
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 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 if necessary 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.

Preparation of the Test Vessels in the second study
The second study was carried out with 6 concentrations within the range 0.18 - 1000 mg a.i./L test item in a geometrical series and with a dilution factor of 5.6. No pH adjustment was made.
All concentrations were pipetted from a stock solution in the Erlenmeyer flasks.
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 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 if necessary 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.

Preparation of the Test Vessels in the third study
The third study was carried out with 5 concentrations within the range 0.025 - 100 mg a.i./L test item in a geometrical series and with a dilution factor of 7.95. No pH adjustment was made.
All concentrations were pipetted from a stock solution in the Erlenmeyer flasks.
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 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 if necessary 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

Remarks:

Copper (II) sulphate pentahydrate p.a.

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: first study

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: first study

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

17.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: first study

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 0.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: second study

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

< 0.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: second study

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

13.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: second study

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

0.025 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: thirs study

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

0.113 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other:

Remarks:

third study

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

13.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: third study

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 test item and reference item was calculated by sigmoidal response and 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 and Specific Respiration Rates of the Control Replicates

For the first study

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	6.54	32.5	20.4
	2	7.02	32.0	20.1
	3	6.76	31.4	19.7
	4	6.80	28.4	17.9
	5	6.59	27.6	17.4
	6	6.62	27.9	17.5
	Mean value		30	18.8
	Standard deviation		2.04
	CV [%]		6.8

*) determined at start of the measurement

Repl. = Replicate

CV = Coefficient of variation

For the second study:

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	7.40	30.3	19.4
	2	7.32	30.2	19.4
	3	7.35	30.4	19.5
	4	7.27	30.8	19.7
	5	7.38	30.9	19.8
	6	7.79	30.1	19.3
	Mean value		30.5	19.5
	Standard deviation		0.3
	CV [%]		0.98

 For the third study:

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	7.12	26.4	19.3
	2	7.22	27.4	20.0
	3	7.39	28.3	20.7
	4	7.28	28.8	21.0
	5	7.36	28.1	20.5
	6	7.58	27.6	20.1
	Mean value		27.8	20.3
	Standard deviation		0.76
	CV [%]		2.7

Oxygen Uptake Rates and Inhibition of the Respiration of the Test Item Concentrations in the first study

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg a.i./L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1	1	7.16	24.2	19	24
	2	6.87	23.5	22
	3	6.85	22.5	25
	4	7.39	23.5	22
	5	5.73	19.9	34
5.6	1	7.86	19.4	35	37
	2	7.27	19.4	35
	3	6.87	18.9	37
	4	7.46	20.1	33
	5	6.57	17.2	43
32	1	7.67	13.1	56	59
	2	7.82	12.6	58
	3	7.32	12.8	57
	4	7.45	12.8	57
	5	6.68	10.5	65
180	1	8.05	3.5	88	89
	2	8.10	3.2	89
	3	7.77	3.3	89
	4	8.11	3.4	89
	5	6.79	3.0	90
1000	1	8.59	0.0	100	100
	2	8.57	0.1	100
	3	8.11	0.0	100
	4	8.50	0.1	100
	5	7.42	0.4	99

*) determined at start of the measurement

Repl. = Replicate

Oxygen Uptake Rates and Inhibition of the Respiration of the Test Item Concentrations in the second study

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg a.i./L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
0.18	1	7.14	26.5	13	13
	2	7.56	26.7	12
	3	7.68	27.0	11
	4	7.78	26.0	15
	5	6.81	26.2	14
1	1	6.54	21.9	28	28
	2	7.35	22.1	28
	3	7.72	22.5	26
	4	7.70	21.5	30
	5	7.13	22.1	28
5.6	1	7.89	19.1	37	37
	2	7.63	19.7	35
	3	7.82	19.2	37
	4	7.59	18.4	40
	5	8.22	19.3	37
32	1	8.13	12.4	59	60
	2	7.91	12.2	60
	3	7.93	12.6	59
	4	7.67	12.2	60
	5	8.06	12.0	61
180	1	8.12	3.1	90	90
	2	8.25	2.9	90
	3	8.36	3.0	90
	4	8.26	2.9	90
	5	8.43	3.0	90
1000	1	8.90	0.0	100	100
	2	8.85	0.4	99
	3	8.98	0.1	100
	4	8.74	0.1	100
	5	8.98	0.1	100

Oxygen Uptake Rates and Inhibition of the Respiration of the Test Item Concentrations in the third study

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg a.i./L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
0.025	1	7.08	24.3	13	3
	2	7.03	28.2	-1
	3	7.07	27.8	0
	4	6.61	25.5	8
	5	7.27	29.0	-4
0.2	1	7.18	24.9	10	13
	2	7.53	23.2	17
	3	7.36	24.8	11
	4	6.74	24.5	12
	5	7.66	23.9	14
1.6	1	7.71	19.5	30	29
	2	7.55	19.8	29
	3	7.62	20.5	26
	4	7.60	18.8	32
	5	7.93	20.5	26
13	1	7.93	14.0	50	49
	2	7.44	14.5	48
	3	7.22	14.0	50
	4	7.76	13.8	50
	5	7.90	14.1	49
100	1	7.85	4.0	86	81
	2	7.54	7.0	75
	3	6.83	7.8	72
	4	7.46	3.5	87
	5	8.05	3.9	86

Validity criteria fulfilled:

yes

Conclusions:

The NOEC in the first study of Basic Blue 159 Trichlorozinkate is < 1 mg a.i./L.
The EC10 < 1 mg a.i./L.
The EC50 is 17.8 mg a.i./L.

The NOEC in the second study of Basic Blue 159 Trichlorozinkate is < 0.18 mg a.i./L.
The EC10 is < 0.18 mg a.i./L.
The EC50 is 13.7 mg a.i./L.

The NOEC of Basic Blue 159 Trichlorozinkate is 0.025 mg a.i./L.
The EC10 is 0.113 mg a.i./L.
The EC50 is 13.1 mg a.i./L.
As this is the most sensitive study, it will be used for further assessment (key values).

Executive summary:

Three Respiration Inhibition Tests with activated sludge according to OECD Guideline No. 209 were carried out with the test item Basic Blue 159 Trichlorozinkate from 2017 -10 -20 to 2017 -12 -13 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 test item concentrations 1 - 5.6 - 32 - 180 - 1000 mg a.i./L, 0.18 - 1 - 5.6 - 32 - 180 - 1000 mg a.i./L and 0.025 - 0.2 - 1.6 - 13 - 100 mg a.i./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.

Remark: The study was performed three times. In the first two studies, no NOEC or EC₁₀ were achieved, but both studies were valid. Therefore, all data and results are given. The results of the third study, as given in the summary, are considered to be the result of the GLP – Study 170329DB / BBR17883. Anyway, the EC-values obtained in all three studies show consistent ranges when compared, underlining the quality of the data.

 

The mean inhibition of respiration for the test item replicates ranged from 3 % to 81 %.

 

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 106 mg/L.

 

NOEC and EC-Values with Confidence Interval of Basic Blue 159 Trichlorozinkateafter 3 h exposure time, Inhibition of Total Respiration

		EC-values [mg a.i./L]		Confidence interval P = 95 [%] [mg a.i./L]
NOEC*		0.025		̶
EC10		0.113		0.060 – 0.207
EC20		0.580		0.343 – 0.927
EC50		13.1		9.59 – 18.5
EC80		94.2		76.2 – > 100

*) No statistically significant inhibition (P < 0.001), ANOVA, Dunnett`s Method

 

 

 

The NOEC of Basic Blue 159 Trichlorozinkate is 0.025 mg a.i./L. The EC10 is 0.113 mg a.i./L and the EC50 is 13.1 mg a.i./L.

 

Description of key information

The NOEC of Basic Blue 159 trichloro zincate is 0.025 mg a.i./L.

The EC10 is 0.113 mg a.i./L.

The EC50 is 13.1 mg a.i./L.

The same values are considered for the target substance (Basic Blue 159 methyl sulfate salt form)

Key value for chemical safety assessment

EC50 for microorganisms:

13.1 mg/L

EC10 or NOEC for microorganisms:

0.025 mg/L