Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Toxicity to microorganisms

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

Two deviations from the study plan and the test guidelines was realized.
1. The time for oxygen content measurement was lower than 8 minutes.
2. The start value of oxygen content for measurement of oxygen consumption was lower than 7 mg/L.
An influence to the results of this study can be excluded.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

304-519-6

EC Name:

Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

94276-33-2

Molecular formula:

C16 H12 Cr N4 O9 S . C11 H25 N O . H

IUPAC Name:

hydrogen 3-[(2-ethylhexyl)oxy]propan-1-amine 11-methyl-4-nitro-12-(phenylcarbamoyl)-6-sulfonato-8λ³-oxa-10λ³-oxa-1λ⁴,13-diaza-9-chromatricyclo[7.4.0.0²,⁷]trideca-1(13),2(7),3,5,10-pentaene-9,9,9-tris(ylium)-8,12-diid-9-olate

Test material form:

solid

Details on test material:

Physical state / Appearance: Solid / orange to brown

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Application of the test substance: Direct addition by initial weight into the test flasks. The test substance was weighed in the required amounts for the test concentrations directly to the test vessels.
- Reference substance preparations: 1000.0 mg of the reference substance was added to about 900 mL of deionized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.2 with 1 M sodium hydroxide solution. Following this the stock solution was made up to 1L with deionized water. The stock solution appeared yellowish and no undissolved reference substance was visible.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

TEST SYSTEM
Age: 1 day
Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
Collection of the test system: 14 Dec 2016
Arrival in the test facility: 14 Dec 2016
After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L Dw.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

21.0 – 21.8°C

pH:

7.5 - 8.3

Dissolved oxygen:

Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 mg/L with the exceptions described below

Nominal and measured concentrations:

1000, 500, 250, 125, 62.5 mg/L as nominal concentration based on test substance without correction of purity and blank controls.
100, 10, 1 mg/L as nominal concentration based on reference substance.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glas-beakers (nominal volume 1L)
- Test volume: 500 mL:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates):
- No. of vessels per reference substance (replicates): 2
- Nitrification inhibitor used (delete if not applicable): none / N-allylthiourea
- Sludge concentration in the test vessel: 1.5 g/L Dw

PREPARATION OF THE TEST
The test substance was added in the required amounts according to the test concentrations directly to the test vessels with 234 mL deionized water. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked. An adjustment was not necessary. After addition of 250 mL of inoculum suspension (3 g/L DW) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance. After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The temperature was measured for seven times in a separate vessel filled with aerated deionized water during incubation phase.
The content of oxygen at the start of the measurements was > 7 mg/L (see below). The oxygen uptake was measured for a period of about 8 to 10 (see below).
The total oxygen consumption of was measured in the sequence BC1-3 and RS1, RS2-5, RS6 and TS1-3, TS4-7, TS8-11 and TS12-15. The oxygen consumption of the blank control BC4-6 were measured at last. No abiotic control was tested.

TEST MEDIUM / WATER PARAMETERS
16 mL/test vessel of 100-fold concentrated OECD medium

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Reason for selection of test concentrations: According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of ≤ 3.2 should be used, preferably encompassing the range from 10 – 80% inhibition.

EVALUATION
The consumption rate (mg O2/L*x minutes) of each test concentration and the blank controls were determined after 3 hours of exposure. The consumption rates (mg O2/L*h) were used to calculate the inhibition effects at particular test concentrations compared to the mean consumption rate of all controls.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 2

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 3

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

Effect concentrations 4

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Validity criteria:
The coefficient of variation of the six replicates of blank control was 16.5 % O2 consumption.
The EC50 of 3,5-dichlorophenol was 4.8 mg/L
The mean oxygen uptake of the blank controls was 19 mg/g*h

The results in this study were consistent with the validity criteria with one exception. The mean oxygen uptake in the blank controls was lower than 20 mg/g*h (19 mg/g*h). Because the reference substance shows an EC50 in the specified range (usual range of EC50 of the last 20 values (within the values in this study) in the laboratory was 3.5 – 18.8 mg/L) and the measured oxygen uptake from the test substance concentrations showed a good curve progression the study is classified as valid. The used activated sludge which was collected from the same waste water treatment plant showed a maximum oxygen consumption of 29.8 mg/g*h and minimum oxygen consumption of 8.2 mg/g*h of the last 20 measurements (within the values in this
study). For this the study is classified as valid.

Results with reference substance (positive control):

In order to verify that the microorganisms are responding normally to toxic stress, tests with a reference substance are conducted according to OECD 209 guideline. The results from the reference substance test are compared to EC50 values published in OECD 209 guideline, which represent the typical response range.
According to the test guideline OECD 209 the EC50 values of the reference substance 3,5-dichlorophenol should be in the range of 2-25 mg/L after 3 hours of incubation.
The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2-25 mg/L in 3 hours.
These results indicate that the microorganisms are responding normally to toxicant stress.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of the degradation activity of activated sludge is not anticipated when introduced to biological treatment plants in appropriate low concentrations