rac-(1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

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:

18 Aug 2014 to 20 Aug 2014

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item amounts of 5.16, 50.24, 500.0, 500.3 and 500.3 mg were weighed on an analytical balance and transferred to the designated test vessels with 234 mL water. The test item was mixed into the water by intense stirring for 15 minutes at room temperature. The pH of the test dispersions was adjusted with a 0.1M NaOH solution from pH 6.5 to the range 7.3 to 7.5.
- Controls: Blank control, containing an equal volume of activated sludge and synthetic medium but no test or reference item, and positive control
- Evidence of undissolved material: All test concentrations were clearly above the water solubility limit of the test item under the test conditions

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
- Preparation of inoculum for exposure: Based on the wet to dry weight ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per litre. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed* per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again and adjusted. The pH of the activated sludge inoculum was adjusted to 7.3.
* Synthetic sewage feed:
16 g peptone;
11 g meat extract;
3 g urea;
0.7 g NaCl;
0.4 g CaCl2.2H2O;
0.2 g MgSO4.7H2O;
2.8 g K2HPO4
Made up to 1 litre with deionised water.
- Pretreatment: None
- Initial biomass concentration: 3 g/L dry weight in the inoculum (equivalent to 1.5 g dry material per litre test medium)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None reported

Hardness:

Not reported

Test temperature:

20 ºC

pH:

Test start range: 7.2 to 7.4
Test end range: 7.7 to 8.0

Dissolved oxygen:

Test start range: 7.6 to 7.9 mg O2/L
Test end range: 8.0 to 8.3 mg O2/L

Nominal and measured concentrations:

- Test substance nominal concentrations: 10, 100 and 1000 mg/L
- Reference substance nominal concentrations: 3.2, 10 and 32 mg/L

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: 2000 mL glass beakers filled with 500 mL incubation mixture
- Aeration: Continuous aeration by intense stirring with magnetic stirrer
- No. of vessels per concentration (replicates): Single replicates for 10 and 100 mg/L test item concentrations and 3 replicates for 1000 mg/L test item concentration
- No. of vessels per control (replicates): 4
- No. of vessels per reference item (replicates): Single replicates
- Sludge concentration (weight of dry solids per volume): 3 g/L dry weight in the inoculum (equivalent to 1.5 g dry material per litre of test medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Distilled or deionised water, containing less than 1 mg/L dissolved organic carbon

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test dispersions was adjusted with a 0.1M NaOH solution from pH 6.5 to the range 7.3 to 7.5
- Light intensity: Normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): pH values and dissolved oxygen concentrations were determined in all test media and the controls at the start and at the end of the 3-hour incubation period. The test media temperature was measured in one control at the start and at the end of the incubation period. For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours of incubation time, and was not further aerated. Subsequently, the dissolved oxygen concentration was measured with an oxygen electrode and was continuously recorded. During measurement, the samples were continuously stirred by magnetic stirrers.

TEST CONCENTRATIONS
- Range finding study: A definitive test was not necessary since in the range-finding test the test item showed no inhibition of the respiration rate at the highest tested concentration (performed as triplicate)

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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

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

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC10

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:

other: EC20

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:

- Effect concentrations exceeding solubility of substance in test medium: Based on the physical appearance of the test suspensions, the saturation concentration was reached at a concentration of <10 mg/L
- Blank controls oxygen uptake rate: The specific respiration rates for both blank control replicates prepared at the beginning of the exposure period were 39 mg O2 per dry weight of activated sludge per hour and for the two replicates prepared at the end of the test, 43 and 41 mg O2/gh
- Coefficient of variation of oxygen uptake rate in control replicates: 5 %

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 10 mg/L (the 95 % confidence limits could not be calculated for mathematical reasons). The 3-hour EC50 is within the guideline-recommended range of 2 - 25 mg/L, confirming suitability of the activated sludge used.

Reported statistics and error estimates:

The 3-hour EC10, EC20, EC50 and EC80 values and their 95 % confidence limits could not be calculated because of the absence of a toxic effect of the test item on the respiration rate of activated sludge at the tested concentrations. The NOEC was derived directly from the raw data without use of statistics. The 3-hour EC50 of the reference item was derived by Probit Analysis using linear maximum likelihood regression.

Table 1. Influence of the Test Item and 3,5-Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge

Vessel No.	Nominal concentration of test chemical (mg/L)	Oxygen comsumption rate		Inhibition (%)	pH values		Oxygen concentration (mg O2/L)
		Respiration rate (R) (mg O2/Lh)	Specific respiration rate (Rs) (mg O2/gh)		start*	end*	start*	end*
Inoculum control
1	0	58.40	38.93		7.3	7.7	7.7	8.0
2	0	58.92	39.28		7.3	7.8	7.8	8.2
11	0	64.50	43.00		7.4	7.9	7.8	8.2
12	0	61.60	41.07		7.4	7.9	7.7	8.2
Mean		60.86	40.57
Standard Deviation		2.81	1.87
Coefficient of Variation		4.61 %	4.61 %
Test item
6	10.0	66.80	44.53	-9.8	7.3	8.0	7.8	8.2
7	100	62.06	41.37	-2.0	7.3	7.8	7.7	8.3
8, 9, 10	1000, 1000, 1000	65.85, 65.73, 64.20	43.90, 43.82, 42.80	-8.2. -8.0. -5.5; Mean = -7.2	7.2, 7.2, 7.4	7.8, 7.9, 7.9	7.6, 7.8, 7.9	8.2, 8.3, 8.2
3,5 -Dichlorophenol
3	3.2	44.95	29.96	26.1	7.3	7.9	7.8	8.2
4	10	35.13	23.42	42.3	7.3	7.9	7.7	8.2
5	32	6.90	4.60	88.7	7.2	7.9	7.7	8.2

* start and end of the 3 -hour incubation period

-% inhibition denotes increased oxygen consumption rate relative to control

Validity criteria fulfilled:

yes

Remarks:

The specific respiration rate of the blank controls was higher than 20 mg O2/gh, the coefficient of variation of oxygen uptake rate in control replicates was less than 30 % and the 3-hour EC50 of 3,5-dichlorophenol is within the range of 2-25 mg/L.

Conclusions:

The 3-hour NOEC value for the inhibitory effect of the test material on the respiration rate of aerobic activated sludge microorganisms was 1000 mg/L, based on nominal concentrations. The 3-hour EC10, EC20, EC50 and EC80 values were determined to be > 1000 mg/L.

Executive summary:

The inhibitory effect of the test item on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209 (2010). A range-finding test was performed with nominal test item concentrations of 10.0, 100 and 1000 mg/L, alongside blank controls and a positive control. The 3-hour NOEC was determined to be 1000 mg/L, the highest rate tested. The 3-hour EC10, EC20, EC50 and EC80 could not be calculated, but were greater than 1000 mg/L. A definitive test was not necessary since in the range-finding test the test item showed no inhibition of the respiration rate at the highest tested concentration. The test concentrations were clearly above the water solubility of the test material under the test conditions at concentrations above 10 mg/L.

Description of key information

3 -h EC50 > 1000 mg/L and NOEC = 1000 mg/L, freshwater (activated sludge of a predominantly domestic sewage), OECD 209, Eisner 2014.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The inhibitory effect of the test item on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209 (2010). A range-finding test was performed with nominal test item concentrations of 10.0, 100 and 1000 mg/L, alongside blank controls and a positive control. The 3-hour NOEC was determined to be 1000 mg/L, the highest rate tested. The 3-hour EC10, EC20, EC50 and EC80 could not be calculated, but were greater than 1000 mg/L. A definitive test was not necessary since in the range-finding test the test item showed no inhibition of the respiration rate at the highest tested concentration. The test concentrations were clearly above the water solubility of the test material under the test conditions at concentrations above 10 mg/L.