N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)azo]-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:

From 17 AUG 2005 to 19 AUG 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

Reference substance used: copper (II) sulphate pentahydrate instead of 3,5-dichlorophenol. The usage of dichlorphenol is unsuitable for routine lab use.

GLP compliance:

yes (incl. QA statement)

Remarks:

according to § 19b Abs. 1 German Chemikaliengesetz and directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test item was weighed out directly into an Erlenmeyer flask,
filled with 250 mL autoclaved tap water and stirred for 24 h.
Dispersion treatment: Agitation
Test concentration: Limit-test at 1000 mg/L

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

TEST SYSTEM: Activated sludge
Reason for the selection of study system: Non adapted activated sludge from the sewage plant at Sarstedt is well suited as it comprises mostly municipal sewage and very little industrial waste water.
Origin: Municipal sewage treatment plant of D-31157 Sarstedt, Germany
Pretreatment: The sludge was washed twice with autoclaved tap water and adjusted corresponding to a dry weight of 4.0 g/L +/- 10 %.
Date of receipt: 2005-08-18
Dry sludge concentration: 4.15 g/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20.6 deg C

pH:

6.75

Dissolved oxygen:

See table on measurements under "any other information on results including tables"!

Nominal and measured concentrations:

Limit test at nominal 1000 mg/L submission substance

Details on test conditions:

2 control samples without test item were included, one at the beginning and one at the end of the test.
TEST METHOD: Static
Test duration 3 h
Replicates: One for limit concentration, reference item concentrations and two for controls
Test vessels: 500 mL Erlenmeyer flasks, DIN 12380
Volume of the study medium: 500 mL
Nutrient solution: Synthetic waste water according to OECD 209

Application:
200 mL inoculum
16 mL synthetic waste water
284 mL autoclaved tap water
Test item concentration (directly weighing)

Test temperature: 18 - 22 °C
Ventilation: Permanent

TYPE AND FREQUENCY OF MEASUREMENT and OBSERVATIONS:
The pH value of the activated sludge was measured prior to testing and the temperature in the measuring cell once during the measurement. The oxygen depletion of the 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. The room temperature during the test was recorded continuously by a hygrothermograph.

COURSE OF THE STUDY:
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 adjusted 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.

EVALUATION:
Determined are the distances recorded by a flat bed recorder (adjusted to 500 mV and 1 cm/min in y-direction) during 3 min measurement for each test vessel.
5 mm on the recorder (x-direction) are equivalent to a decrease of 0.1 mg O2/L. That leads to the factor f = 0.02 mg O2/L x mm.
The respiration rate (R) according to the raw data was calculated in the following way:
R = md x f x t
R : respiration rate mgO2/Lxh
md : measured distance mm/3min
f : 0.02 mgO2/Lxmm
t : 20 [3min/h]
The inhibition of respiration rates were calculated by a standard method as outlined in OECD 209.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Key result

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:

After stirring for 24 h the test item was mostly dispersed.
For further details see section "Any other information on results including tables".

Results with reference substance (positive control):

REFERENCE ITEM Copper (II) sulphate pentahydrate p.a. MERCK
CAS RN 7758-99-8
Lot A265990
Purity 99.9 %
Expiry date 2005-11-30
Test concentrations 58 - 100 - 180 mg/L
Range of validity (EC50) 51 - 157 mg/L

In the reference test the EC50 -value for copper(II)-sulphate-pentahydrate was determined by probit analysis with:
EC50 = 99 mg/L
This is in the recommended confidence range of 51 - 157 mg/L. The range of validity (EC50 +/- 3SD) of copper (II) sulfate pentahydrate is calculated from the mean of all reference tests (n=238) from 1991 - 2005.

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).

Raw Data and Respiration Rates of Test Item, Control and Reference Item

	Concentration [mg/L]	Oxygen- concentration* [mgO2/L]	Measured distance** [mm/3min]	Respiration rate [mgO2/L¿h]
Test item	1000	6.64	78	31.2
Control 1	-	6.16	86	34.4
Control 2	-	7.74	76	30.4
Reference item	180	8.15	17	6.8
	100	8.60	42	16.8
	58	6.94	60	24.0

 

* determined at the start of the 3 minute measurement

** 5 mm are related to 0.1 mgO2/L

Respiration Rates and Inhibition of Respiration of Test and Reference ItemConcentrations

	Concentration [mg/L]	Respiration rate [mgO2/L¿h]	Inhibition [%]
Test item	1000	31.2	4
Control 1	-	34.4	-
Control 2	-	30.4	-
Reference item	180	6.8	79
	100	16.8	48
	58	24.0	26

 Difference of the control respiration rates: 12 % Promotion of respiration is signed with minus (-)

Validity criteria fulfilled:

yes

Conclusions:

In a respiration inhibition test with activated sludge according to OECD 209 no respiration inhibition was observed at the nominal test item concentration of 1000 mg/L (limit test).
EC50 (3h) > 1000 mg/L (nominal)
NOEC (3h) = 1000 mg/L (nominal)

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 and compliant to GLP (reliability category 1) was performed with the submission substance. Test system was activated sludge of a municipal treatment plant. The test was performed under static conditions with the nominal limit concentration 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 99 mg/L. Validity criteria of the test guideline were fulfilled. The submission substance is not toxic at concentrations up to 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Description of key information

Key study: Respiration inhibition test with activated sludge according to OECD 209. No respiration inhibition was observed at the nominal test item concentration of 1000 mg/L (limit test). EC50 (3h) > 1000 mg/L (nominal). NOEC (3h) = 1000 mg/L (nominal)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L