2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.03,19.05,17.06,14.07,12.023,25.024,32.025,30]hexatriaconta-1(36),2,5,7,9,11,14,16,18,20,23,25,27,29,32,34-hexadecaene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study according to OECD Guideline 209

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)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION

Preparation of the saturated solution: The test item was weighed out (1000 mg/L ) directly into an Erlenmeyer flask and filled up with 250 mL autoclaved tap water. It was stirred for 24 h.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Origin: Municipal sewage treatment plant of D-31157 Sarstedt
- non-adapted activated sludge, receives predominatly municipal sewage and hardly industrial chemical waste
- Preatreatment: the sludge was washed twice with autoclaved tap water and adjusted corresponding to a dry weight of 4.0 g/L +/- 10 %

Test type:

static

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

A range-finding test (Non-GLP-state) was carried out with the concentrations: 10 - 100 - 1000 mg/L). Based on the results of the preliminary test the study was carried out with the limit test concentration of 1000 mg/L.

Test temperature:

18 - 22°C

pH:

6,86

Dissolved oxygen:

7.25 mg/L

Nominal and measured concentrations:

Nominal concentration: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Erlenmeyer flasks, DIN 12380
- fill volume: 500 mL
- Aeration: permanent
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM
- 200 mL inoculum
- 16 mL synthetic waste water
- 284 mL autoclaved tap water
- test item concentration (directly weighing)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- the pH-value of the activated sludge was measured prior to testing
- the temperature in the measuring cell was determined once durcing the measurement
- the room temperature during the test was recorded continously by a hygrothermograph
TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10 mg/L, 100 mg/L, 1000 mg/L
- Results used to determine the conditions for the definitive study: based on the results of the preliminary test the study was carried out with a
limit test concentration of 1000 mg/L. Two control replicates without test item were included.

Reference substance (positive control):

yes

Remarks:

Copper-II-sulphate pentahydrate p.a. (Merck)

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:

EC0

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:

pH-value of the activated sludge: 6.86
dry sludge concentration: 4.24 g/L
test temperature: 21.1°C

Results with reference substance (positive control):

In the reference test the EC50-value for copper(II)-sulphate-pentahydrate was calculated by probit analysis usiing software SIGMAPLOT.
EC50 (3 h) = 113 mg/L
The two control respiration rates differ not more than 15 % in the study.
The EC50 (3 h) of copper(II)-sulphate-pentahydrate is in the accekpted range of 53 - 157 mg/L.

Results of the range finding test:

Table 1: Inhibitions of the range-finding test

Concentration [mg/L]	Inhibition [%]
10	-2
100	-8
1000	-16

Respiration rates and inhibition of respiration

From the oxygen depletion the respiration rates were calculated in mg O₂/L x h. 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 2: Respiration rates and inhibition of respiration of test and reference item concentrations

	Concentration [mg/L]	Respiration Rate [mg O2/L x h]	Inhibition [%]
Test item	1000	38.8	- 17
Control 1	-	34.4	-
Control 2	-	32.0	-
Reference item	180	8.8	73
Reference item	100	18.0	46
Reference item	58	27.6	17

Table 3: Raw data and respiration rates of test item, control and reference item

	Concentration [mg/L]	O2 Concentration[mg O2 /L]	Measured Distance[mm/3 min]	Respiration Rate [mg O2/L x h]
Test item	1000	7.25	97	38.8
Control 1	-	6.39	86	34.4
Control 2	-	6.74	80	32.0
Reference item	180	8.07	22	8.8
Reference item	100	7.63	45	18.0
Reference item	58	7.39	69	27.6

Validity criteria fulfilled:

yes

Remarks:

The EC50-value for the reference item was 113 mg/L.

Conclusions:

The test item is not toxic at concentration

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was performed with the test item (batch-no. DEB 2060256) 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 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 of the test item was observed at the tested 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 113 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.

Description of key information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was performed with the test item. 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.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L