6,8-Dioxabicyclo[3.2.1]octan-4-one, (1S,5R)-

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:

07 - 16 Apr 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)

Remarks:

Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland

Specific details on test material used for the study:

Reference Item:
The reference item and the following information were supplied by Acros Organics BVBA/Belgium:

Identity: 3,5-Dichlorophenol
Product No.: 11361
Batch No.: A0323035
Purity: 99.6% (GC)
Expiration Date: 30 July 2017 (retest date)
Storage Conditions (as provided by the supplier): Store in a cool, dry place, in a tightly closed container.
Storage Conditions (as handled at Harlan Laboratories): At room temperature at 20 ± 5°C, in the dark.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Procedure: At the start of the test, 16 mL synthetic sewage were made up to 250 mL with water and then 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were added to this first control vessel. Thereafter, in time intervals of about fifteen minutes (an arbitrary but convenient interval) the procedure was repeated, except that the 16 mL synthetic sewage were added to the appropriate amounts of reference item or test item in water to obtain a volume of 250 mL, followed by the addition of 250 mL inoculum. The addition of the inoculum to the incubation mixture is considered as the start of the 3-hour incubation time period.

Dosage: Based on the results of the range-finding test, the test item was soluble at a concentration of at least 5 g/L in tap water. A stock solution with a nominal concentration of 5 g/L was prepared by dissolving 5009 mg of the test item in 1000 mL water and thoroughly mixed by intense stirring for 15 minutes. In this way a clear solution was obtained. The pH of the stock solution was adjusted from pH 4.0 to pH 7.4 with a 1M NaOH solution. Adequate volumes of this stock solution were diluted with water to prepare the test media with the lower test item concentrations. Then, 16 mL of synthetic sewage feed and 250 mL of the activated sludge inoculum were added.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: The sludge was washed twice with chlorine free 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. Based on this ratio, a calculated amount of wet sludge was resuspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. 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 readjusted to the level of 3 g dry weight per liter in the inoculum used for the test, equivalent to 1.5 g/L in the final incubation mixture. The pH of the activated sludge inoculum was 7.1.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Nominal and measured concentrations:

Control, 62.5, 125, 250, 500 and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM-

Test vessel:
- Material, size, headspace, fill volume: 2000-mL glass beakers containing 500 mL of incubation mixture
- Aeration: continuous- No. of vessels per concentration (replicates): 3
- No. of vessels per blank control (replicates): 6
- No. of vessels per positive control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Synthetic sewage feed: The synthetic sewage feedwas prepared to contain the following constituents per liter filled up to the final volume with deionized water: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 K2HPO4The pH of this solution was pH 7.3.

- Test water: Distilled or deionized water, containing less than 1 mg/L dissolved organic carbon was used except where chlorine free tap water is specified.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate, 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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Results used to determine the conditions for the definitive study: The test item was soluble at a concentration of at least 5 g/L in tap water.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

555 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: 289 - >1000 mg/L

Key result

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

Remarks on result:

other: 95% CL: 782 - >1000 mg/L

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

Remarks on result:

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

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

The 3 hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 9 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 2–25 mg/L, confirming the suitability of the activated sludge used.

Reported statistics and error estimates:

The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration using suitable software (ToxRat Professional).The NOEC was derived by per-vessel statistics using the Dunnett t-test (Dunnett, 1955; Dunnett, 1964) (one sided smaller, α= 0.05). The 3-hour EC10, EC20, EC50 and EC80 values and their 95% confidence limits were, as far as possible, derived by Probit Analysis (Davies, 1971; Finney, 1971).The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Probit Analysis(Davies, 1971; Finney, 1971).DAVIES, R.G. (1971): Computer programming in quantitative biology. Academic Press, London. FINNEY, D.J. (1971): Probit analysis,3rd edition. University Press, London. DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. Journal of the American Statistical Association, 50, pp. 1096–1121.DUNNETT, C.W. (1964): New tables for multiple comparisons with a control. Biometrics, 20, pp. 482–491.

Respiration Rates of the Blank Controls:

The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 47 and 44 mg O₂ per dry weight of activated sludge per hour, for the two blank controls prepared after the reference item group were 50 and 45 mg O₂/gh and for the two replicates prepared at the end of the test were 45 and 44 mg O₂/gh. All six values are higher than the 20 mg O2/gh stipulated in the guideline. The resulting coefficient of variation of oxygen uptake rate in the control replicates has a value of 5% and is therefore, as stipulated in the guideline, not more than 30%.

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

Vessel No.	[mg/L] Nominal concentration of test chemical	Oxygen consumption 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*
Control
1	0	70.53	47.02			7.1	7.8	8.1	8.4
2	0	65.47	43.64			7.1	7.8	8.1	8.6
6	0	74.53	49.69			7.0	7.9	8.6	9.6
7	0	66.84	44.56			7.0	7.9	8.5	8.4
23	0	66.93	44.62			7.1	7.8	8.1	8.3
24	0	66.00	44.00			7.2	7.8	8.3	8.6
Mean		68.38	45.59
Standard Deviation		3.50	2.33
Coefficient of Variation		5.11%	5.11%
Test Item					Mean
8	62.5	72.67	48.44	-6.3	-3.4	7.1	7.9	8.0	8.5
9	62.5	67.05	44.70	2.0		7.2	7.8	8.4	8.5
10	62.5	72.48	48.32	-6.0		7.1	7.9	8.0	8.9
11	125	70.40	46.93	-2.9	-5.4	7.1	7.8	7.9	9.0
12	125	74.67	49.78	-9.2		7.1	7.9	8.1	8.6
13	125	71.07	47.38	-3.9		7.2	7.9	8.0	8.7
14	250	76.80	51.20	-12.3	-10.3	7.1	7.8	8.0	8.2
15	250	72.67	48.44	-6.3		7.0	7.9	8.2	8.5
16	250	76.80	51.20	-12.3		7.1	7.9	7.9	8.5
17	500	65.40	43.60	4.4	6.2	7.1	7.9	7.9	8.1
18	500	68.04	45.36	0.5		7.2	8.0	8.0	8.6
19	500	59.00	39.33	13.7		7.2	8.0	8.0	9.0
20	1000	56.40	37.60	17.5	17.1	7.2	8.0	8.4	8.6
21	1000	56.22	37.48	17.8		7.1	8.0	8.1	8.8
22	1000	57.43	38.29	16.0		7.1	8.0	7.9	9.0
Reference Item
3	3.2	52.11	34.74	23.8		7.0	7.9	8.0	9.1
4	10	34.07	22.71	50.2		7.0	7.8	8.0	8.9
5	32	9.60	6.40	86.0		7.0	7.7	8.8	9.5

* Start and end of the 3-hour incubation period.

- % inhibition: Increased oxygen consumption rate relative to control.

Validity criteria fulfilled:

yes

Conclusions:

A 3 hour EC50 of >1000 mg/L and NOEC of 500 mg/L were determined for the substance using a relevant test method and in compliance with GLP. The result is considered to be reliable.

Description of key information

Toxicity to microorganisms: ASRI 3-hour EC₅₀ value of >1000 mg/L and NOEC of 500 mg/L (nominal concentrations).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

500 mg/L

Additional information

A 3-hour EC₅₀ value of >1000 mg/L (highest concentration tested) and a NOEC of 500 mg/L (nominal concentrations) have been determined for the effects of the test substance, Cyrene™, on respiration inhibition of activated sludge, in accordance with OECD Test Guideline 209. This indicates that the test substance was not toxic to wastewater (activated sludge) bacteria at a loading rate of 1000 mg/L.

In view of the properties of the substance, it is thought that the organisms were exposed to the Gem Diol form of the substance.