4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxy-N-(2-methylphenyl)naphthalene-2-carboxamide

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

28 APR 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to OECD Guideline 209 compliant to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

Copper (II) sulphate pentahydrate instead of the prescribed 3,5- dichlorophenol as a reference substance. The usage of dichlorophenol is unsuitable for routine lab use.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German "Chemikaliengesetz", EC and OECD Guideline principles

Test material

Test material information

Constituent 1

Reference substance name:

4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-phenylnaphthalene-2-carboxamide

EC Number:

227-930-1

EC Name:

4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-phenylnaphthalene-2-carboxamide

Cas Number:

6041-94-7

IUPAC Name:

4-[(2,5-dichlorophenyl)diazenyl]-3-hydroxy-N-phenyl-2-naphthamide

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was weighed out directly into an Erlenmeyer flask and filled up with 250 mL autoclaved tap water.
- Dispersion treatment: stirring for 24 h
After stirring for 24 h the test item was mostly dispersed. Only part of the test item floated on the surface and stuck to the side of the Erlenmeyer flask.
Test concentration: 1000 mg/L

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Pretreatment: The sludge was washed twice with autoclaved tap water and adjusted corresponding to a dry weight of 4.0 g/l ± 10 %.
- Dry sludge concentration 4.12 g/L

Non adapted activated sludge from the sewage plant at Sarstedt, Germany, is well suited as it comprises mostly municipal sewage and very
little industrial waste water.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

21.0 °C

pH:

6.95 (pH of the activated sludge)

Nominal and measured concentrations:

1000 mg/l nominal, no analytical measurements

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks, DIN 12380
- Fill volume: 500 mL
- Aeration: Permanent
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): two
- Biomass loading rate: 200 ml inoculum/500 ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 284 mL autoclaved tap water and 16 mL synthetic waste water according to OECD 209 per 500 mL flask

OTHER TEST CONDITIONS
The pH-value of the activated sludge was measured prior to testing and the temperature in the measuring cell once during the measurement.
The room temperature during the test was recorded continuously by a hygrothermograph.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen depletion of control, reference and test item was measured after 3 h incubation by means of oximeter and recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1 nominal limit concentration

A range-finding test (NON-GLP-state) was carried out with three concentrations: 10 - 100 - 1000 mg/L.
Based on the results of the preliminary test the study was carried out with a limit test concentration of 1000 mg/L.
2 control replicates without test item were included.
A fresh sample of activated sludge taken from the sewage plant Sarstedt was used for the test procedure. It was washed twice and diluted with autoclaved tap water to a dry sludge concentration of 4.0 g/L ± 10 %.
The reference item was tested in three concentrations within the concentration range 58 - 180 mg/L with a dilution factor of 1.8.
The respiration rates of all samples were determined subsequently to an incubation period of 3 h.

Reference substance (positive control):

yes

Remarks:

copper (II) sulphate pentahydrate, p.a.

Results and discussion

Effect concentrations

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

Results with reference substance (positive control):

In the reference test the EC50 -value for copper(II)-sulphate-pentahydrate was determined by probit analysis with:
EC50 = 107 mg/L
This is in the recommended confidence range of 53 - 155 mg/L.

Reported statistics and error estimates:

The EC50-value of the reference item was calculated with probit analysis using software SIGMAPLOT (Windows) rel. 8.02, 2002, SPSS CORPORATION
Probit values were taken from WEBER (1986).
Confidence intervals were calculated according to a standard procedure (BREITIG & TÜMPLING 1982).

BREITIG, G., TÜMPLING, W. (editors, 1982): Methoden der Wasseruntersuchungen Bd.II. VEB GUSTAV FISCHER VERLAG, Jena.
WEBER, E. (1986): Grundriß der biologischen Statistik, 9. Auflage

Any other information on results incl. tables

Table 1: Respiration rates and inhibition of respiration of test and reference item concentrations

	Concentration [mg/L]	Respiration rate [mgO2/L¿h]	Inhibition [%]
Test item	1000	30.4	6
Control 1	-	33.2	-
Control 2	-	31.6	-
Reference item	180	8.4	74
	100	16.8	48
	58	25.6	21

Difference of the control respiration rates: 5 %

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was performed compliant to GLP with the submission substance (reliability category 1). Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test was performed under static conditions with the nominal limit concentration of 1000 mg/L. The respiration rates of control, reference and test item concentrations were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates. No inhibition was observed at the tested limit concentration.

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 107 mg/L.

Validity criteria of the test guideline were fulfilled.

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