1,4-Dioxaspiro[4.5]decane, 8-[4-(4-methylphenyl)-3-cyclohexen-1-yl]-

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:

2017-11-08 to 2018-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Based on the results of the preliminary test it could concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in 3 replicates (limit test to determine the NOEC). The concentration was weighed out directly in the Erlenmeyer flasks.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Activated sludge, microorganisms from a domestic waste water treatment plant
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant of Hildesheim, Germany
- Pretreatment: The Sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The Sludge was used within 24 h after sampling.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20°C

pH:

pH of activated sludge: 7.49
pH of synthetic waste water: 7.27

Dissolved oxygen:

Control samples : 6.99 - 7.80 mg/O2/L;
Test item sample: 5.72 – 5.74 mg O2/L

Nominal and measured concentrations:

Nominal conc.: 1000 mg/L;
Measured conc.: not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks
- Type: open
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % Saturation and to maintain the Sludge flakes in Suspension.
- No. of vessels per concentration (replicates): main study: 3
- No. of vessels of positive control (replicates): 3
- No. of vessels of control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 2.80 g/L (corresponding to 1.40 g/L suspended solids in the test vessels)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

EFFECT PARAMETERS MEASURED: The oxygen uptake rates of all samples will be determined following an incubation period of 3 h.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate

Key result

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

Results with reference substance (positive control):

Copper-(II)-sulphate pentahydrate
EC50 = 104 mg/L

Validity Criteria

• The mean specific oxygen uptake rate of the control replicates was 20.5 mg O₂/g.h (validity criterion: should be > 20 mg O₂/g.h).


• The coefficient of variation of the oxygen uptake rates in the control replicates was 2.9 % (validity criterion: < 30 %).


• The EC₅₀ of the reference item was 104 mg/L and therefore in the range of 53 - 155 mg/L.

 

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration

Table 1: Oxygen Uptake Rates, Inhibition of the Respiration of Control Replicates

 

	Replicate	Oxygen Concentration* [mg O2/L]	Oxygen Uptake Rate R [mg O2/(L*h)]	Specific Respiration Rate Rs [mg O2/g*h]
Control	1	7.67	28.0	20.0
	2	7.62	27.3	19.5
	3	7.56	28.6	20.4
	4	7.09	29.8	21.3
	5	6.99	29.3	20.9
	6	7.80	29.0	20.7
	Mean value		28.7	20.5
	Standard deviation		0.83
	CV [%]		2.9

 

Table 2: Oxygen Uptake Rates and Inhibition of the Respiration of the Test Item Concentration

 

Test Item Concentration [mg/L]	Replicate	Oxygen Concentration* [mg O2/L]	Oxygen Uptake Rate R [mg O2/(Lxh)]	Inhibition [%]	Mean Inhibition [%]
1000	1	5.74	29.2	-2
	2	5.81	28.9	-1	-1
	3	5.72	28.9	-1

*) determined at Start of the measurement

CV = Coefficient of Variation

 

Table 3: NOEC of the test item

 

	Test item Concentration [mg/L]
NOEC*	> 1000

 

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item is ≥ 1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.

Executive summary:

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions in a study according to OECD TG 209. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of activated sludge was measured in the presence of various concentrations of the test item after an incubation period of 3hours. The test was carried out under static conditions with the limit test item 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. The mean inhibition of respiration for the test item replicates was -1%. 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 104 mg/L. The validity criteria of the test guideline were fulfilled. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L. Thus, the NOEC of the test item is determined to ≥ 1000 mg/L.

Description of key information

The NOEC of the test item is ≥ 1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L (reference 6.1.7-1).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions in a study according to OECD TG 209. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of activated sludge was measured in the presence of various concentrations of the test item after an incubation period of 3hours. The test was carried out under static conditions with the limit test item 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. The mean inhibition of respiration for the test item replicates was -1%. 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 104 mg/L. The validity criteria of the test guideline were fulfilled. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L. Thus, the NOEC of the test item is determined to ≥ 1000 mg/L.