Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

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:

2014

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

Specific details on test material used for the study:

Name: FAT 20033/L
Batch number: AT-PD13-359A1
EAS Test item code: 2014-000179
Appearance / colour: solid / dark blue
Date of analysis: 07 Jan 2014
Expiry date: 07 Jan 2019
Storage conditions: ambient (+5 to +30 °C), dark, dry

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant of 75172 Pforzheim, Germany, collected from the aeration tank, was used as the microbial inoculum for the test. This plant was predominantly treating domestic sewage.

Test type:

static

Water media type:

not specified

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

the test assays were aerated during the whole test period

Hardness:

no data

Test temperature:

19.2 – 21.4 °C (measured during O2-determination at 3 h after start of respiration)

pH:

7.28 – 7.88

Dissolved oxygen:

Oxygen content in the control after 3 h (total oxygen uptake assay)
3.96 to 7.25 mg O2/L

Nominal and measured concentrations:

no data

Details on test conditions:

Two litre glass beakers were used; the test volume was 500 mL; the test period was 3h; 16 mL synthetic sewage feed was used;

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol (DCP)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

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:

The total oxygen uptake was measured up to a test item concentration of 1000 mg/L.
After 3 hours, an inhibitory effect of -11.3 % was determined in the highest test item concentration. For the heterotrophic oxygen uptake, after 3 hours, an average inhibitory effect of 2.8 % was determined at the highest test item concentration. For the oxygen uptake due to nitrification an average inhibitory effect of -117.7 % was determined at the highest test item concentration after 3 hours.
Due to the fact that there was no inhibition at the highest test item concentration compared to the control, the NOEC was determined to be 1000 mg/L.

Results with reference substance (positive control):

The EC50 of the total oxygen uptake for the reference item DCP was estimated between 5-25 mg/L after 3 hours. This is within the accepted range of 2 to 25 mg/L for total respiration. For the heterotrophic oxygen uptake for DCP, the EC50 was estimated between 5-25 mg/L after 3 hours. This is within the recommended range of 5 to 40 mg/L. For the oxygen uptake due to nitrification the EC50 was estimated between 0.2-1 mg/L after 3 hours. This value was also within the recommended range of 0.1 to 10 mg/L.

Reported statistics and error estimates:

The statistical evaluation was performed for specific respiration rate of the control and the highest test item concentrations using SAS® Software service pack 9.3 (2002–2010). The control and 1000 mg/L was statistically tested, using the pooled t-test (left sided).

Results, specific respiration rates

	Specific respiration rates [mg O2/(g × h)]
	3 h	3h	3h
Test assay	Total	Heterotrophic	Due to nitrification
Control 1	23.49	20.51
Control 2	21.22	18.95	2.6*
Control 3	21.69	19.14
10 mg/L	23.55	16.92	6.63
100 mg/L	21.19	19.55	1.64
1000 mg/L 1	23.23	18.15
1000 mg/L 2	25.09	19.96	5.66*
1000 mg/L 3	25.59	18.84
DCP 0.2 mg/L	21.17	17.27	3.90
DCP 1.0 mg/L	15.81	15.16	0.65
DCP 5.0 mg/L	13.94	15.04	-1.10
DCP 25.0	2.61	2.75	-0.14
Abiotic Control 1	0.43
Abiotic Control 2	0.02
Abiotic Control 3	0.48

*these results are the difference between the mean of total specific respiration rate and the mean of heterotrophic respiration rate

Results, inhibition

Test assay nominal [mg/L]	Inhibition [%]
	Total	Heterotrophic	Due to nitrification
	3 h	3 h	3 h
10	-6.4	13.4	-155.0
100	4.2	-0.1	36.9
1000 (mean)	-11.3	2.8	-117.7
DCP 0.2	4.3	11.6	-50 .0
DCP 1.0	28.6	22.4	75.0
DCP 5.0	37.0	23.0	142.3
DCP 25.0	88.2	85.9	105.4

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of FAT 20033/L was determined to be greater than 1000 mg/L and the NOEC was determined to be 1000 mg/L.

Executive summary:

The respiration rate of an activated sludge, fed with a standard amount of synthetic sewage feed, was measured 3 hours after application of the test item. The inhibitory effects of FAT 20033/L in particular concentrations were expressed as a percentage of the mean respiration rate of the controls. This test was conducted according to OECD guideline 209. Activated sludge from the municipal wastewater treatment plant of Pforzheim, Germany, was used as microbial inoculum for the test. This plant predominantly is treating domestic sewage. A range-finding test was performed with concentrations from 10, 100 and 1000 mg/L in a limit test design. The highest concentration of 1000 mg/L and a control was tested in three replicates whereas 10 and 100 mg/L were tested in one replicate. Four concentrations of 3,5 - dichlorophenol (DCP) as toxic reference item were tested in parallel to demonstrate the sensitivity of the test system. To test the differences between total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrification, two test assays were performed, one with and one without allylthiourea (ATU) respectively. The oxygen uptake rate for the range-finding test was determined after 3 hours. The total oxygen uptake was measured up to a test item concentration of 1000 mg/L. After 3 hours, an inhibitory effect of -11.3 % was determined in the highest test item concentration. For the heterotrophic oxygen uptake, after 3 hours, an average inhibitory effect of 2.8 % was determined at the highest test item concentration. For the oxygen uptake due to nitrification an average inhibitory effect of -117.7 % was determined at the highest test item concentration after 3 hours. Due to the fact that there was no inhibition at the highest test item concentration compared to the control, the NOEC was determined to be 1000 mg/L.The test item FAT 20033/L had no toxic effect on the oxygen uptake at the highest concentration tested (1000 mg/L). Hence, the EC₅₀was determined to be > 1000 mg/L. All validity criteria of OECD Guideline for Testing of Chemicals No. 209 were fulfilled:

•  The mean oxygen uptake rate of the blank control were determined to be 22 mg O₂/(g × h) dry matter after 3 hours. Hence the rate was not less than 20 mg oxygen uptake per g of activated sludge (dry weight of suspended solids) per hour.

•  The coefficients of variation of the control for total oxygen uptake were 5% and for the heterotrophic oxygen uptake 4 % after 3 hours and did not exceed 30 % at the end of the test.

• The EC50 of the total oxygen uptake for DCP was estimated between 5-25 mg/L after 3 hours.This is within the accepted range of 2 to 25 mg/L for total respiration. For the heterotrophic oxygen uptake for DCP, the EC50was estimated between 5-25 mg/L after 3 hours. This is within the recommended range of 5 to 40 mg/L. For the oxygen uptake due to nitrification the EC50was estimated between 0.2-1 mg/L after 3 hours. This value was also within the recommended range of 0.1 to 10 mg/L.

 

In conclusion; The EC50 of FAT 20033/L was determined to be greater than 1000 mg/L and the NOEC was determined to be 1000 mg/L.

Description of key information

The EC50 of FAT 20033/L was determined to be greater than 1000 mg/L and the NOEC was determined to be 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The effect of the test item on microorganisms of an aerobic microbial treatment plant was tested using the activated sludge respiration inhibition test. The test item FAT 20033/L had no toxic effects on the oxygen uptake at the highest concentration tested (1000 mg/L). Hence, the EC50 of FAT 20033/L was > 1000 mg/L and the NOEC is 1000 mg/L. All validity criteria according to OECD Guideline for Testing Chemicals 209 were fulfilled. Several supporting studies investigating toxicity of Acid Blue 277 to bacterial population also indicate IC50 to be >300 mg/L, thereby supporting the conclusion that the substance has low toxicity to microbes.