2-[(2-methoxy-4-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

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:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study meets requirements of OECD Guideline 209 and GLP requirements with only minor deviations (see below).

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

In contrast to the guideline, it was decided to use copper (II) sulphate pentahydrate instead of the prescribed 3,5-dichlorophenol. The usage of dichlorophenol is unsuitable for routine lab use.

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)
- Method: The test substance was weighed out directly into an Erlenmeyer flask, filled up with 250 mL autoclaved tap water and stirred for 24 h
- Differential loading: 1000 mg/L
- Controls: 2 control samples without test substance were included

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 4.0 g/L +/- 10 %
- Dry sludge concentration: 3.69 g/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

21.7 °C

pH:

6.61

Salinity:

Not determined

Nominal and measured concentrations:

Nominal concentration: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Composition of test medium: 200 mL inoculum + 16 mL synthetic waste water (acc. to the guideline) + 284 mL autoclaved tap water + test substance concentration (directly weighing of 500 mg)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Determination of the respiration rates subsequently to an incubation period of 3 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test concentration
- Range finding study
- Test concentrations: 10 - 100 - 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes
Table 1: Inhibitions of the Range-Finding Test
Concentration[mg/L] Inhibition
[%]
10 0
100 -3
1000 -1
Promotion of respiration is signed with minus (-)

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: Before stirring the test substance was insoluble. After stirring the test substance was partly dispersed, floated on the surface and stuck to the side of the flask.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 109 (CI 54 - 156) mg/L

Reported statistics and error estimates:

The EC50 of the reference item was calculated with probit analysis using software SIGMAPLOT (Windows), SPSS CORPORATION.
Probit values were taken from WEBER (1986).

From the oxygen depletion the respiration rates were calculated in mg O₂/L×h (Table 2).

The inhibition of the respiration rates from the test and reference item concentrations in % was calculated in comparison with the respiration rates from the control.

Table 1:    Respiration Rates and Inhibition of Respiration of Test and Reference Item Concentrations

	Concentration	Respiration rate	Inhibition
	[mg/L]	[mgO2/L×h]	[%]
Test item	1000	28.8	-1
Control 1	–	29.2	–
Control 2	–	27.6	–
Reference item	180	7.2	75
	100	15.6	45
	58	22.8	20

Difference of the control respiration rates: 5 %                 Promotion of respiration is signed with minus (-)

Validity criteria fulfilled:

yes

Conclusions:

No inhibition was observed for the test item. The tes item is not toxic at concentrations

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item on 2008-10-21 at Dr.U.Noack-Laboratorien, D-31157 Sarstedt. Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test was carried out under static conditions with the limit 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. No inhibition was observed for the test item.

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 109 mg/L.

Validity criteria of the test guideline were fulfilled.

Description of key information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out as a limit test. The test was performed under static conditions with the nominal limit concentration of 1000 mg/L.

The test item is not toxic at concentrations ≤ 1000 mg/L to activated sludge of a municipal sewage treatment plant.