(1R,4S,4aR,8aS)-9-(dichloromethylidene)octahydro-1,4-methanonaphthalene-5,8-dione

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:

28 February 2011 to 10 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

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: No stock solution was made due to low water solubility of the test item. 5.09, 16.06, 50.42, 160.59 and 500.16 mg of test substance were weighed by means of an analytical balance and transferred to the designated test vessels with 284 mL tap water. The test item was mixed into the tap water by ultrasonic treatment for fifteen minutes and intense stirring for three hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible.
- Eluate: Tap water.
- Controls: Two controls of tap water containing synthetic sewage feed and inoculum. Reference item 3, 5-dichlorophenol was tested at three concentrations in parallel under identical conditions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Appearance of test media showed that all test concentrations were clearly above the water solubility limit of the test material.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
- Method of cultivation: Aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated:
- 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 three 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 in the inoculum used for the test. The pH of the activated sludge inoculum was 7.5.
* Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl filled up to a final volume of 1 liter
0.4 g CaCl2 × 2H2O with deionized water
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4
Made up to 1 litre with deionized water

- Pretreatment: None
- Initial biomass concentration: 3.6 g/L dry weight (corresponding to about 1.4 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:

19ºC to 21ºC

pH:

7.7 to 8.4

Dissolved oxygen:

7.5 mg/L to 8.5 mg/L after 3-hour incubation period and just before respiration rate measurements

Salinity:

Not applicable

Nominal and measured concentrations:

Test substance nominal concentrations: 10, 32, 100, 320 and 1000 mg/L.
Reference substance nominal concentrations: 5, 16 and 50 mg 3, 5-dichlorophenol/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2000 ml glass beakers
- Material, size, headspace, fill volume: See table below
- Aeration: Continuous aeration by intense stirring with magnetic stirrer.
- Sludge concentration: 3.6 g/L dry weight (corresponding to about 1.4 g dry material per litre of test medium).
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per reference item(replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No. Culture medium utilized tap water and also dieonized water (50ml per day) in synthetic sewage feed.
- Intervals of water quality measurement: Beginning and end of 3 hour incubation period.

OTHER TEST CONDITIONS
- Adjustment of pH: No. pH measured at start and end of 3 hour incubation period.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen respiration rate measured continuously after 3 hour incubation period (see below for more details).

Reference substance (positive control):

yes

Remarks:

3, 5-Dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

343 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL 105 - 498 mg/L.. NOEC determined as the calculated 3-hour EC10.

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

427 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL 172 - 586 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

650 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL 413 - 857 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

991 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL 755 - > 1000 mg/L

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: All test concentrations were clearly above the water solubility of the test material under the test conditions.

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 12 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.

Reported statistics and error estimates:

The 3-hour EC values of the test and reference item and their 95%-confidence limits were, as far as possible, calculated by Probit Analysis

Table 1. Influence of 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 consumption rate (mg O2L^-1min^-1)	Inhibition (%)	PH values		Oxygen Concentration (mg O2L^-1)
				start*	end*	start*	end*
Control
A	0	1.449		7.7	8.4	8.1	7.9
B	0	1.355		7.7	8.2	8.0	7.9
Mean		1.402
Deviation (%)		6.5
Test Item
4	10	1.36	3	7.9	8.1	8.1	7.7
5	32	1.443	-2.9	8.0	8.2	8.2	7.8
6	100	1.28	8.7	7.9	8.2	8.1	8.0
7	320	1.293	7.8	7.9	8.2	8.1	8.0
8	1000	0.273	80.5	7.9	8.3	8.2	8.4
Reference item
1	5	1.271	9.3	7.8	8.4	8.0	7.5
2	16	0.467	66.7	7.7	8.3	8.0	7.9
3	50	0.146	89.6	7.8	8.2	8.2	8.5
* start and end of the 3-hour incubation period
-% inhibition: increased oxygen consumption rate relative to control

Validity criteria fulfilled:

yes

Remarks:

Yes. (The oxygen consumption rates of the two controls is within 15% of each other and the 3-hour EC50 of the reference substance is within the guideline-recommended range of 5 - 30 mg 3, 5-dichlorophenol/L).

Conclusions:

The calculated 3-hour EC50 and NOEC values for the inhibitory effect of the test material on the respiration rate of aerobic activated sludge microorganisms were 650 (95% CL: 413 - 857) and 343 (95% CL; 105 - 498) mg/L respectively, based on nominal concentrations. All test concentrations were clearly above the water solubility of the test material under the test conditions.

Executive summary:

The sensitivity of activated sludge microorganisms to the test material was determined in a GLP-compliant test performed to standard guidelines. The 3-hour EC50 was found to be 650 mg/L with 95 % confidence limits of 413 - 857 mg/L. The 3-hour NOEC (determined as the calculated 3-hour EC10 value) was found to be 343 mg/L with 95 % confidence limits of 105 – 498 mg/L. All results are based on nominal concentrations and test concentrations were clearly above the water solubility of the test material under the test conditions.

Description of key information

3-h EC50 and 3-h NOEC were 650 (95% CL: 413 - 857) and and 343 (95% CL; 105 - 498) mg/L respectively, based on nominal concentrations, OECD 209, static, activated sludge of a predominantly domestic sewage, Eisner 2011

All test concentrations were clearly above the water solubility of the test material under the test conditions.

Key value for chemical safety assessment

EC50 for microorganisms:

650 mg/L

EC10 or NOEC for microorganisms:

343 mg/L

Additional information

The sensitivity of activated sludge microorganisms to the test material was determined in a GLP-compliant test performed to standard guidelines. Five concentrations of test item were tested, namely 10, 32, 100, 320 and 1000mg/l. The 3h EC50 was claculated to be 650mg/l based on nominal concentrations. The result is considered reliable and adequate for risk assessment, classification and labelling.