2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge nitrification inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.12.1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 9509 (Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms)

Version / remarks:

First edition 1989-10-15

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge

Details on inoculum:

The study was performed with activated sludge, cultivated in a model activated sludge plant in the laboratories of RCC. The cultivation of sludge was not performed in compliance with the GLP Regulations and these data are not mentioned in the report.

Preparation of inoculum:
Before use in the test, the sludge was washed twice by settling the sludge, discarding the supernatant liquid and resuspending the sludge in medium. Then the sludge was homogeneously suspended in an appropriate volume of medium to give the
required concentration of mixed liquor suspended solids (MLSS) of about 3 g dry weight/L in the inoculum. The exact concentration of solids in this inoculum was determined by filtering a part of this inoculum through a GF/C glass microfibre filter with a pore size of about 1.2 μm (produced by Whatman, England) and weighing the dried residue.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

4 h

Test temperature:

21.7 - 21.9 °C at the start and 22.9 -23.6 °C at the end of the test

pH:

8.0 - 8.8

Dissolved oxygen:

> 7.7 mg/l

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg test substance/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 500 ml, -, 250 ml
- Aeration: no
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): n.a.
- No. of vessels per abiotic control (replicates): n.a.
- Sludge concentration (weight of dry solids per volume): 3 g dry weight/L
- Nutrients provided for bacteria: no
- Nitrification inhibitor used (delete if not applicable): allylthiourea (ATU control)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: n.a. due to short test duration of 4 h
- Light intensity: diffused light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration inhibiation
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: no
- Test concentrations: 10, 32, 100, 320, 1000 mg/L

Reference substance (positive control):

yes

Remarks:

Allylthiourea

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

673 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

4 h

Dose descriptor:

EC10

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 100% inhibition

Reported statistics and error estimates:

- 4-hour EC50: 673 mg test substance/L (95 % conf. limits: 245 - 14751 mg test substance/L)
- 4-hour EC10: 21 mg test substance/L (95 % conf. limits: 0 - 65 mg test substance/L)
- 4-hour EC90: > 1000 mg test substance/L

The measured amounts of formed nitrate nitrogen, the calculated nitrification rates and the percentage inhibition of formed nitrate nitrogen after 4 hours incubation period are listed in Table 1. The nitrification rate in the control amounted to 2.0 mg N/(g x h). The calculated nitrification rate is based on the amount of formed nitrate nitrogen. The amount of formed nitrite nitrogen was not measured (in deviation to the ISO Guideline), since an incomplete oxidation of ammonium only to nitrite, but not to nitrate, is not desirable and indicates a partial inhibition of nitrification.

In the ATU-control no nitrate nitrogen has been formed. The calculated inhibition rate of the test item on the nitrification of activated sludge micro-organisms after the incubation period of 4 hours amounted to:

- 4-hour EC₅₀: 673 mg test substance/L (95 % conf. limits: 245 - 14751 mg test substance/L)

- 4-hour EC₁₀: 21 mg test substance/L (95 % conf. limits: 0 - 65 mg test substance/L)

- 4-hour EC₉₀: > 1000 mg test substance/L

All reported results are related to the nominal concentrations of the test substance.

Table 1: Amounts of formed nitrate nitrogen, the calculated nitrification rates and the percentage inhibition of formed nitrate nitrogen after 4 hours incubation period

Nominal concentration of test substance (mg/L)	Formation of nitrate nitrogen  (mg NO3-N/(L x 4h))	Nitrification rate* (mg N/(g x h))	%-Inhibition of formed nitrate nitrogen
Control	9.3	2.0	/
ATU Control	0.0	/	/
10	8.4	1.8	9.7
32	8.3	1.8	10.8
100	7.9	1.7	15.1
320	5.0	1.1	46.2
1000	4.1	0.9	55.9

*measured dry weight of activated sludge in inoculum (MLSS): 2.3 g/l

Validity criteria fulfilled:

yes

Conclusions:

The 4-h EC10 and EC50 values based on the inhibition of the nitrification rate of the test sample to microorganisms is 21 mg/L and 673 mg/L, respectively.

Executive summary:

The inhibitory effect on the nitrification rate of the test item to microorganisms originating from originating from activated sludge, cultivated in a model activated sludge plant in the laboratories of RCC was investigated after 4 h of exposure at nominal concentrations of 0 (control), 10, 32, 100, 320, 1000 mg/L. In the ATU-control no nitrate nitrogen has been formed.

The calculated inhibition rate of the test item on the nitrification of activated sludge micro-organisms after the incubation period of 4 hours amounted to:

- 4-hour EC₅₀: 673 mg test substance/L (95 % conf. limits: 245 - 14751 mg test substance/l)

- 4-hour EC₁₀: 21 mg test substance/L (95 % conf. limits: 0 - 65 mg test substance/l)

- 4-hour EC₉₀: > 1000 mg test substance/L

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18.07.1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

April 4, 1984, corresponding to ETAD-Method No.103

Deviations:

yes

Remarks:

see field "Principles of method if other than guideline"

Principles of method if other than guideline:

Modification from the guideline: Sludge from a Husman laboratory apparatus, higher microbial inoculum concentration, higher synthetic sewage feed concentration, and higher temperature variation

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

other: mixed culture of bacteria (2.0 g/L) (sludge of a Husman apparatus)

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

3 h

Test temperature:

22.3 °C

Nominal and measured concentrations:

1 / 3.2 / 10 / 32 / 100 mg/L (nominal)

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: the IC50 (3h) of the reference substance 3,5-Dichlorophenol is: 16.5 mg/L (Criteria: 10-25 mg/L)

Table 1: Bacteria inhibition calculated on basis of the oxygen consumption after 3h

Test substance (mg/L)	O2 consumption (mg/L/min)	Inhibition (%)
100	1.08	32.92
32	1.38	14.29
10	1.42	11.80
3.2	1.46	9.32
1	1.68	0
Control 1	1.69	mean: 1.61
Control 2	1.52

Validity criteria fulfilled:

yes

Conclusions:

The estimated IC50 (3h) of the test sample to microorganisms originating from a mixed culture of bacteria (2.0 g/L) (sludge of a Husman apparatus) is above 100 mg/L.

Executive summary:

The inhibitory effect of the test item to microorganisms originating from a mixed culture of bacteria (2.0 g/L) (sludge of a Husman apparatus) was investigated after 3 h of exposure at nominal concentrations of 0 (control), 1, 3.2, 10, 32, and 100 mg/L. The reference substance (positive control) 3,5-dichlorophenol was tested at concentrations of 10, 32, and 100 mg/L with a total exposure duration of 3 h. Results with the reference substance were valid.

The estimated IC50 (3 h) of the test sample to microorganisms is above 100 mg/L.

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11.01.1993 - 14.05.93

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: activated sludge from a water treatment works treating mainly domestic sewage
- Name and location of sewage treatment plant where inoculum was collected: not mentioned
- Method of cultivation: date of sludge batch and date of test are the same - no cultivation; test conditions: 20.8°C
- Preparation of inoculum for exposure: no
- Pretreatment: no
- Initial biomass concentration: not given

Test type:

not specified

Water media type:

freshwater

Remarks:

dechlorinated mains water

Limit test:

no

Total exposure duration:

30 min

Remarks on exposure duration:

Cationic substances adsorb strongly, leading to a rapid decrease of the substance concentration in aqueous media in the presence of sewage sludge, justifying a shorter exposure duration of 30 min.

Test temperature:

20.8 °C

Nominal and measured concentrations:

nominal: 0 (control) / 10 / 32 / 100 / 320 / 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1-litre glass beakers placed in a water bath to maintain a temperature of 20°C
- Material, size, headspace, fill volume: The test beakers are prepared containing synthetic sludge feed, the appropriate volume of stock solution, and dilution water to a linal volume of 300 ml. A volume of 200 ml diluted sludge is then added, the beaker placed in the water bath and aerated continuously at a constant rate for the duration of the test.
- Aeration: Aeration of the test beakers is started at suitable time intervals, to allow for the oxygen measurement
- No. of vessels per concentration (replicates): n/a
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 4 g solid per litre

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated mains water

OTHER TEST CONDITIONS
- Details on termination of incubation:
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : At the end of the exposure period the sludge suspension from each beaker is transferred rapidly into a standard BOD bottle which is then placed on a magnetic stirrer and a DO probe inserted. The dissolved oxygen concentration of the suspension is recorded continuously using a chart recorder for a period of up to 10 minutes. The rate of respiration is calculated from the printout of the recorder, over a range
when the plot is linear.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca 3.2
- Range finding study: no

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Key result

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

1 064 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: estimated

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: estimated EC 50 (3h) = 11 mg/L
- Other: estimated EC20 (3h) = 4 mg/L

Table 1: Respiration rates of activated sludge exposed to the test item for 30 minutes

Concentration of sample (mg/L)	Respiration rate (mgO2/L.hr)	Inhibition (%)
0 (control)	mean 43.2	-
10	42.0	2.8
32	40.8	5.6
100	41.0	5.1
320	33.6	22.2
1000	19.8	54.2

From these results the EC₅₀ value (30 minutes) is estimated to be 1064 mg/litre. In terms of orders of magnitude, this is expressed as 1,000 to 10,000 mg/I.

The present experimental results suggest that the sample tested shows a significant inhibitory effect on the respiration rate of activated sludge after a contact period of 30 minutes. The estimated EC₅₀ value for the sample was 1,064 mg/L, which is slightly higher than the highest concentration tested. As recommended by the OECD protocol, this may be expressed in orders of magnitude as 1,000 to 10,000 mg/L.

Dilution of the test solution from 1,000 mg/l to 100 mg/l had a significant effect on the degree of inhibition. The % inhibition of the activated sludge respiration rate decreased from 54.2 % to 5.1 % over this range of test sample concentrations. Significant inhibition was not considered to occur until a concentration of 186 mg/l was reached (the EC₂₀ value). It was not considered necessary to carry out the test at higher concentrations because 1 g/L was the highest concentration expected to be present in the environment.

Validity criteria fulfilled:

not specified

Conclusions:

The estimated EC50 (30 min) of the test sample to microorganisms originating from predominantly domestic sewage is 1064.0 mg/L.

Executive summary:

The inhibitory effect of the test item to microorganisms originating from sewage sludge of a STP treating predominantly domestic sewage was investigated after 30 min of exposure at nominal concentrations of 0 (control), 10, 32, 100, 320 and 1,000 mg/L. The reference substance (positive control) 3,5-dichlorophenol was tested at concentrations of 0 (control), 5, 16 and 50 mg/L with a total exposure duration of 3 h. Results with the reference substance were valid.

The present experimental results suggest that the sample tested shows a significant inhibitory effect on the respiration rate of activated sludge after a contact period of 30 minutes. The estimated EC₅₀value for the sample was 1064.0 mg/L, which in terms of orders of magnitude (as recommended by the OECD protocol) is expressed as 1,000 to 10,000 mg/L.

Dilution of the test solution from 1,000 mg/L to 100 mg/L had a significant effect on the degree of inhibition. The % inhibition of the activated sludge respiration rate decreased from 54.2 % to 5.1 % over this range of test sample concentrations. Significant inhibition was not considered to occur until a concentration of 186 mg/l was reached (the EC₂₀ value).

Considering the actual concentration of the active ingredient in the test sample of 58 - 60%, the estimated EC₅₀ value (30 min) for the active ingredient can be calculated as ca. 617 - 638 mg/L.

Reference 4

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26.02.1990

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

not specified

Analytical monitoring:

not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Nominal and measured concentrations:

Nominal: 0 (control), 10, 32, 100 mg/L

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Table 1: Bacteria inhibition calculated on basis of the oxygen consumption after 3h

Test substance (mg/L)	O2consumption (mg/L/min)	Inhibition (%)
100	0.57	64.1
32	0.93	41.5
10	1.21	23.9
Control 1	1.54	mean: 1.59
Control 2	1.63

3,5 Dichlorophenol (mg/L)	O2consumption (mg/L/min)	Inhibition (%)
100	0.09	94.3
32	0.24	84.9
10	1.14	28.9

Validity criteria fulfilled:

not specified

Conclusions:

The EC50 (3 h) of the test sample to microorganisms under the conditions of the OECD test guideline 209 is 50 mg/L.

Executive summary:

The inhibitory effect of the test item to microorganisms under the conditions of the OECD test guideline 209 was investigated after 3 h of exposure at nominal concentrations of 0 (control), 10, 32 and 100 mg/L. The reference substance (positive control) 3,5-dichlorophenol was tested at the same concentrations. Results with the reference substance were valid.

The present experimental results suggest that the sample tested shows a significant inhibitory effect on the respiration rate of microorganisms after a contact period of 3 h. The estimated EC₅₀value for the sample was 50 mg/L.

Reference 5

Endpoint:

activated sludge nitrification inhibition testing

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

673 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

4 h

Dose descriptor:

EC10

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 100% inhibition

Reported statistics and error estimates:

- 4-hour EC50: 673 mg test substance/L (95 % conf. limits: 245 - 14751 mg test substance/L)
- 4-hour EC10: 21 mg test substance/L (95 % conf. limits: 0 - 65 mg test substance/L)
- 4-hour EC90: > 1000 mg test substance/L

The measured amounts of formed nitrate nitrogen, the calculated nitrification rates and the percentage inhibition of formed nitrate nitrogen after 4 hours incubation period are listed in Table 1. The nitrification rate in the control amounted to 2.0 mg N/(g x h). The calculated nitrification rate is based on the amount of formed nitrate nitrogen. The amount of formed nitrite nitrogen was not measured (in deviation to the ISO Guideline), since an incomplete oxidation of ammonium only to nitrite, but not to nitrate, is not desirable and indicates a partial inhibition of nitrification.

In the ATU-control no nitrate nitrogen has been formed. The calculated inhibition rate of the test item on the nitrification of activated sludge micro-organisms after the incubation period of 4 hours amounted to:

- 4-hour EC₅₀: 673 mg test substance/l (95 % conf. limits: 245 - 14751 mg test substance/l)

- 4-hour EC₁₀: 21 mg test substance/l (95 % conf. limits: 0 - 65 mg test substance/l)

- 4-hour EC₉₀: > 1000 mg test substance/l

All reported results are related to the nominal concentrations of the test substance.

Table 1: Amounts of formed nitrate nitrogen, the calculated nitrification rates and the percentage inhibition of formed nitrate nitrogen after 4 hours incubation period

Nominal concentration of test substance (mg/L)	Formation of nitrate nitrogen  (mg NO3-N/(L x 4h))	Nitrification rate* (mg N/(g x h))	%-Inhibition of formed nitrate nitrogen
Control	9.3	2.0	/
ATU Control	0.0	/	/
10	8.4	1.8	9.7
32	8.3	1.8	10.8
100	7.9	1.7	15.1
320	5.0	1.1	46.2
1000	4.1	0.9	55.9

*measured dry weight of activated sludge in inoculum (MLSS): 2.3 g/l

Validity criteria fulfilled:

yes

Conclusions:

The 4-h EC10 and EC50 values based on the inhibition of the nitrification rate of the test sample to microorganisms is 21 mg/L and 673 mg/L, respectively.

Executive summary:

The inhibitory effect on the nitrification rate of the test item to microorganisms originating from originating from activated sludge, cultivated in a model activated sludge plant in the laboratories of RCC was investigated after 4 h of exposure at nominal concentrations of 0 (control), 10, 32, 100, 320, 1000 mg/L. In the ATU-control no nitrate nitrogen has been formed.

The calculated inhibition rate of the test item on the nitrification of activated sludge micro-organisms after the incubation period of 4 hours amounted to:

- 4-hour EC₅₀: 673 mg test substance/L (95 % conf. limits: 245 - 14751 mg test substance/L)

- 4-hour EC₁₀: 21 mg test substance/L (95 % conf. limits: 0 - 65 mg test substance/L)

- 4-hour EC₉₀: > 1000 mg test substance/L

This information is used in a read-across approach in the assessment of the target substance.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reference 6

Endpoint:

activated sludge respiration inhibition testing

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: the IC50 (3h) of the reference substance 3,5-Dichlorophenol is: 16.5 mg/L (Criteria: 10-25 mg/L)

Table 1: Bacteria inhibition calculated on basis of the oxygen consumption after 3h

Test substance (mg/L)	O2 consumption (mg/L/min)	Inhibition (%)
100	1.08	32.92
32	1.38	14.29
10	1.42	11.80
3.2	1.46	9.32
1	1.68	0
Control 1	1.69	mean: 1.61
Control 2	1.52

Validity criteria fulfilled:

yes

Conclusions:

The estimated IC50 (3h) of the test sample to microorganisms originating from a mixed culture of bacteria (2.0 g/L) (sludge of a Husman apparatus) is above 100 mg/L.

Executive summary:

The inhibitory effect of the test item to microorganisms originating from a mixed culture of bacteria (2.0 g/L) (sludge of a Husman apparatus) was investigated after 3 h of exposure at nominal concentrations of 0 (control), 1, 3.2, 10, 32, and 100 mg/L. The reference substance (positive control) 3,5-dichlorophenol was tested at concentrations of 10, 32, and 100 mg/L with a total exposure duration of 3 h. Results with the reference substance were valid.

The estimated IC₅₀ (3 h) of the test sample to microorganisms is above 100 mg/L.

This information is used in a read-across approach in the assessment of the target substance.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reference 7

Endpoint:

activated sludge respiration inhibition testing

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

1 064 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: estimated

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: estimated EC50 (3h) = 11 mg/L
- Other: estimated EC20 (3h) = 4 mg/L

Table 1: Respiration rates of activated sludge exposed to the test item for 30 minutes

Concentration of sample (mg/L)	Respiration rate (mgO2/L.hr)	Inhibition (%)
0 (control)	mean 43.2	-
10	42.0	2.8
32	40.8	5.6
100	41.0	5.1
320	33.6	22.2
1000	19.8	54.2

From these results the EC50 value (30 minutes) is estimated to be 1064 mg/litre. In terms of orders of magnitude, this is expressed as 1000 to 10 000 mg/I.

The present experimental results suggest that the sample tested shows a significant inhibitory effect on the respiration rate of activated sludge after a contact period of 30 minutes. The estimated EC50 value for the sample was 1,064 mg/L, which is slightly higher than the highest concentration tested. As recommended by the OECD protocol, this may be expressed in orders of magnitude as 1,000 to 10,000 mg/L.

Dilution of the test solution from 1,000 mg/L to 100 mg/L had a significant effect on the degree of inhibition. The % inhibition of the activated sludge respiration rate decreased from 54.2 % to 5.1 % over this range of test sample concentrations. Significant inhibition was not considered to occur until a concentration of 186 mg/l was reached (the EC20 value). It was not considered necessary to carry out the test at higher concentrations because 1 g/L was the highest concentration expected to be present in the environment.

Validity criteria fulfilled:

not specified

Conclusions:

The estimated EC50 (30 min) of the test sample to microorganisms originating from predominantly domestic sewage is 1064.0 mg/L.

Executive summary:

The inhibitory effect of the test item to microorganisms originating from sewage sludge of a STP treating predominantly domestic sewage was investigated after 30 min of exposure at nominal concentrations of 0 (control), 10, 32, 100, 320 and 1,000 mg/L. The reference substance (positive control) 3,5-dichlorophenol was tested at concentrations of 0 (control), 5, 16 and 50 mg/L with a total exposure duration of 3 h. Results with the reference substance were valid.

The present experimental results suggest that the sample tested shows a significant inhibitory effect on the respiration rate of activated sludge after a contact period of 30 minutes. The estimated EC₅₀ value for the sample was 1064.0 mg/L, which in terms of orders of magnitude (as recommended by the OECD protocol) is expressed as 1,000 to 10,000 mg/L.

Dilution of the test solution from 1,000 mg/L to 100 mg/L had a significant effect on the degree of inhibition. The % inhibition of the activated sludge respiration rate decreased from 54.2 % to 5.1 % over this range of test sample concentrations. Significant inhibition was not considered to occur until a concentration of 186 mg/l was reached (the EC₂₀ value).

Considering the actual concentration of the active ingredient in the test sample of 58 - 60%, the estimated EC₅₀value (30 min) for the active ingredient can be calculated as ca. 617 - 638 mg/L.

This information is used in a read-across approach in the assessment of the target substance.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reference 8

Endpoint:

toxicity to microorganisms, other

Type of information:

not specified

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

other: ETAD 3002

Version / remarks:

Ecological And Toxicological Association Of Dyes and Organic Pigments Manufacturers (ETAD)

Deviations:

not specified

GLP compliance:

not specified

Test organisms (species):

Escherichia coli

Dose descriptor:

IC50

Effect conc.:

> 100 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

not specified

Validity criteria fulfilled:

not specified

Conclusions:

According to company data the IC50 for the test item concerning the growth inhibition of Escherichia coli was determined to be above 100 mg/L.

Executive summary:

According to company data the IC₅₀ for the test item concerning the growth inhibition of Escherichia coli was determined to be above 100 mg/L. The test was conducted in accordance with method 3002 from the  Ecological And Toxicological Association Of Dyes and Organic Pigments Manufacturers (ETAD).

Description of key information

Three studies rated with Klimisch 1 or 2 are available in the assessment of the endpoint “Toxicity to microorganisms”. These studies were conducted with structural analogues of the target substance.

Memmert (1993) has been rated as Klimisch 1 and is thus identified as key study. This study investigates the inhibition of the nitrification rate of activated sludge after 4-h exposure to different concentrations of the test item of 10, 32, 100, 320, and 1000 mg/L. The 4-h EC₁₀ and EC₅₀ values were determined with 21 mg/L and 673 mg/L, respectively.

In a supporting study by Smith (1993; Klimisch 2), the inhibitory effect of the respective test substance on the respiration rate of domestic sewage sludge was investigated after 30 min of exposure instead of a 3 h exposure period as foreseen by the current OECD Guideline 209. This shorter exposure period is, however, in line with an older version of OECD 209 that was applicable at the time of testing (OECD 209, version 1984). In addition, cationic substances in particular generally adsorb strongly. This adsorption is linked to a rapid decrease of the test substance concentration in aqueous media in the presence of sewage sludge particles. Thus, a shorter exposure duration of 30 min is justified. Validity of the studies was assured by running respective positive control samples in the tests. A 30-min EC₅₀ values of 1064 mg/L was determined in this study. Taking into account the actual concentration of the active ingredient of 58-60 %, the 30-min EC₅₀ for the pure substance can be calculated as 617-638 mg/L, which is in line with the 4-h EC₅₀ value reported by Memmert (1993).

These values are further supported by information available from Schnalke (1991; Klimisch 2), and Anonymous (1980; Klimisch 4): the 50 % inhibitory concentration for the O₂ respiration of a mixed bacteria culture determined according to OECD 209 after an exposure time of 3 hours was reported to be > 100mg/L (33% inhibition at 100 mg/L). The EC₅₀ for the growth Escherichia coli is > 50 mg/L (based on the active ingredient concentration; method ETAD 3002, exposure duration not reported).

Additional company information (Klimisch 4) is available, which reports results determined in accordance with OECD guideline 209 at lower test substance concentrations (Anonymous, 1990). The 3-h IC₅₀ for the inhibition of the respiration rate was determined with 50 mg/L. Due to the insufficient documentation of the study, it is not possible to determine the cause for the lower effects levels seen in this study.

A possible explanation for the deviation could be the source of the sludge, differing physicochemical parameter (e.g. temperature, pH), or deficiencies in the test performance. Therefore, this study is not further considered in the substance evaluation.

 

The chosen key values of 21 mg/L (4-h EC₁₀) and 673 mg/L (4-h EC₅₀) from Memmert (1993) are based on the test item containing > 90 % active ingredient. These values are considered reliable, robust, and protective enough in a worst-case scenario. Hence, this value is used forward in the risk characterisation of the substance.

Key value for chemical safety assessment

EC50 for microorganisms:

673 mg/L

EC10 or NOEC for microorganisms:

21 mg/L

Additional information

All information except for the data referenced by Anonymous (1980) is used in a read-across approach in the assessment of the target substance Basic Blue 41 methyl sulfate.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.