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

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jul 2014 to 22 Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Haddington Municipal Sewage Works, East Lothian, UK - treating predominantly domestic wastewater
- Storage conditions: under aeration at test conditions
- Storage length: for the duration of the holding period (ca 1 day)
- Preparation of inoculum for exposure: On arrival at the laboratory, as the sludge was sufficiently settled, a portion of the overlying supernatant was siphoned off to concentrate the solid sewage inoculant portion. The resultant sludge was then shaken vigorously by hand to homogenise and the suspended solid content was determined by drying in an oven, duplicate aliquots (5 mL) of the homogenised sludge at approximately 105°C. The suspended solid content was determined as 2.740 g/L. This was outside the required range (3-5 g/L), therefore the sludge was concentrated further by settling for 65 mins prior to ca 1.35 L of the clear overlying supernatant being removed. The sludge was then homogenised by hand and the suspended solid content was then re-determined, as 3.682 g/L. After solid content determination, the sludge was shaken and allowed to settle for 61 mins. Duplicate aliquots (5 mL) of the clear overlying supernatant were taken for suspended solids content determination. The suspended solids content of the supernatant was determined to be 638 mg/L. This was used to calculate the volume of supernatant (86 mL) required to achieve a microbial inoculum of 27.50 mg suspended solids/L in each 2 L bioreactor.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: To obtain the final mineral medium, 10 mL of stock solution A and 1 mL each of stock solution B, C and D were combined and made up to 1000 mL with deionised water. A) KH2PO4 8.50 g/L, K2HPO4 21.75 g/L, Na2HPO4 x 2H2O 33.40 g/L, NH4Cl 0.50 g/L, B) CaCl2 x 2H2O 36.40 g/L, C) MgSO4 x 7H2O 22.50 g/L, D) FeCl3 x 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter
- Test temperature: 21-23 °C, monitored using a max-min thermometer. Temperatures recorded daily
- pH: At test initiation, the pH of each test bioreactor was measured. Each bioreactor was then made up to final volume (2 L). At the end of incubation, the pH was measured again in each test flask on Day 28 prior to acidification and on Day 29 post acidification.
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The test bioreactors (2 L capacity glass flat bottom flasks, labelled with the study number, vessel ID and treatment/test concentration) were incubated under continuous aeration and mixing for the duration of the test (28 Days). Aeration was supplied to the system
by compressed air at a flow rate of 30-55 mL/min. CO2 was removed from the air before entering the test system by filters containing self-indicating soda lime granules (Durasorb). At the end of the test (Day 28), the pH was measured in each bioreactor and then 1 mL of concentrated hydrochloric acid was added. The bioreactors were aerated overnight to drive off the residual carbon dioxide and then all three Ba(OH)2 traps connected to each bioreactor were titrated.
- Number of culture flasks/concentration: 2

SAMPLING
- Sampling frequency: Day 2,3,5,8,10,12,15,18,22,26,29
- Sampling method: CO2 evolution from each test flask was measured by passing the air from the test flask through three 100 mL barium hydroxide (Ba(OH)2) traps. Trap changes were made at regular intervals (1-3 Days for the first 2 weeks and thereafter at 3-5 days) throughout the test. The production of CO2 was determined by titration with 0.05M Hydrochloric acid (HCl) using phenolphthalein indicator.

CONTROL AND BLANK SYSTEM
- Inoculum blank: medium and inoculum
- Toxicity control: contained both the test item and the reference item

STATISTICAL METHODS:
The tabulated values represent rounded results obtained by calculation using the exact raw data. The weight of CO2 evolved was calculated from the titre using the equation below (taken from OECD Guideline 301B (1992)). The background titre was determined for each freshlyprepared barium hydroxide solution. The titre value for each trap was determined and subjected to the following equation: Weight CO2 produced (mg) = 1.1 (background titre – mL HCl titrated). The net CO2 production was calculated by subtracting the mean inoculum control CO2 production values from test item, toxicity control and procedural control CO2 production values. The percentage biodegradation in calculated using the following equation: % biodegradation = (mg CO produced)/(mg TOC added in test x 3.67)x 100%, where 3.67 is the conversion factor (44/12) for carbon to carbon dioxide.

Reference substance:

benzoic acid, sodium salt

Test performance:

No unusual observations or other information that may have affected results.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

< 5

Sampling time:

28 d

Details on results:

See table 1 in "Any other information on results incl. tables"

The test material was not biodegradable under the test conditions within 28 days.

Table 1. Cumulative Biodegradation in the Test Bioreactors of the test item and the procedure control 

Percentage Biodegradation (%)
Time [days]	Test item Replicate No.		Procedure control Replicate No.
	1	2	1	2
2	0.0	0.1	26.3	26.7
3	0.0	0.5	47.2	47.1
5	0.3	1.0	65.3	64.5
8	0.3	1.1	76.4	75.5
10	0.6	1.5	82.6	81.5
12	0.7	1.7	85.4	84.5
15	1.0	1.9	87.6	87.2
18	1.2	1.9	88.6	88.4
22	1.3	1.9	90.2	89.8
26	1.3	1.9	91.0	90.7
28	1.4	2.1	91.2	90.7
Mean (Day 28)	1.8		91.0

 

Validity of the test

The results are considered valid since the following criteria were met:

- The mean CO2 production in the inoculum control (medium and inoculum) was 10.3 mg CO2/L within 28 days (criterion: not greater than 40 mg CO2/L).

- The percentage biodegradation of the reference item reached the level for ready biodegradability by Day 5 (criterion: at least 60% of ThCO2 by Day 14). The mean percent biodegradation at Day 12 was 85.0%.

- The IC content of the test substance suspension in the mineral medium at the beginning of the test was less than 5% (0.44%) of the TC.

Since the test item was not degraded, other validity criteria are not applicable.

Validity criteria fulfilled:

yes

Remarks:

see "Any other information on results incl. tables"

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material was not biodegradable under the conditions of the test.

Executive summary:

The ready biodegradability of test item the test item was investigated at a concentration of 20 mg total organic carbon (TOC) L-1 in a CO2 Evolution (Modified Sturm) Test, OECD 301B, under GLP. Domestic aerobic activated sewage sludge was incubated under continuous aeration, at 21-23°C in the dark over 28 days. The levels of carbon dioxide (CO2) evolved were measured at appropriate intervals throughout the test and these were compared with the theoretical CO2 evolution to determine the extent of biodegradation.

The mean cumulative percentage biodegradation of the test item in the test media at the end of the test (Day 28) was 1.8%. In the toxicity control, containing both test item and the reference item, sodium benzoate, the cumulative percentage biodegradation was greater than 35% (45.5%) based on total TOC. According to the guideline, this indicates that the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration. The cumulative percentage biodegradation in the toxicity control was similar to the procedural controls containing sodium benzoate only (90.7%), if calculated only on the amount of sodium benzoate added. In the procedure controls, the reference item sodium benzoate was degraded by an average of 85.0% by exposure Day 12 and reached an average cumulative percentage biodegradation of 91.0% by the end of the test (Day 28), thus confirming suitability of the activated sludge. In accordance with the guideline, the test item was therefore not ready biodegradable under the test conditions.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Dec 2014 to 20 Jan 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Version / remarks:

1981

Deviations:

yes

Remarks:

Temperature was kept at 20±1°C instead of 25±2°

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge was collected from ten sites recommended by the Guidelines for the Testing of Chemicals, Ministry of Protection Agency of P.R. China, and is one of the recommended test systems of OECD Guidelines for the Testing of Chemicals. Activated sludge is suggested to be derived from the areas where a variety of chemicals are used and discharged. The sources of inoculum used in this study included sewage treatment works, rivers, lakes, etc.
- Fresh samples were collected from ten sites including aeration tank of Shenyang North Sewage Treatment Plant, sewage outfall of Shenshuiwan Sewage Treatment Plant, sewage outfall of Shenyang Xiannvhe Sewage Treatment Plant, Weigong Channel, Maguan Bridge, Hekou Wetland, Nanhu Park, Hunhe River, Xinkai River, Beiling Park, on November 04, 2014. Samples of sludge, surface soil, water, etc. were collected respectively and 3 liters were mixed by stirring in a single container.
- Pretreatment: After floating matter was removed and allowing to stand, the supernatant was filtered through a fine sieve. The pH of the supernatant was determined to be 8.22 and adjusted to be 7.63 with phosphoric acid. 9 L of the filtered supernatant was used to fill two fill-and-draw activated sludge vessel (identified as A and B) respectively and the liquid was aerated overnight. Thirty minutes after stopping aeration, about one third of the whole volume of supernatant was discarded and an equal volume of 0.1 percent synthetic sewage (pH 7.0 ± 1.0) containing 0.1 % each of glucose, peptones and monopotassium phosphate was added to the settled material and re-commence aeration for about 23.5 h. This procedure was repeated once every day. Temperature was kept at 23.0-25.9°C and pH at 7.0 ± 1.0, sludge was sufficiently aerated to keep the mixture aerobic at all times.
- During the culturing step, the following items were checked to estimate the state of the culture on the 10th day after beginning of culturing and then once a week, the results showed that the state of activated sludge was normal:
- Appearance of supernatant: the supernatant of active sludge was clear;
- Precipitability of active sludge: the active sludge, being in large flocks, had high precipitability;
- State of formation of active sludge: the growth of flocks was observed;
- pH: The pH of the supernatant was 6.39- 7.42;
- Amount of aeration: the dissolved oxygen concentration of the solution was 5.38 - 6.73 ppm when determination, which was above 5 ppm;
- Microflora of activated sludge: When the active sludge was microscopically observed, a number of protozoa of different species together with cloudy flocks were seen.
- Preparation of inoculum for exposure: After 46 days in culture, i.e., before three days of test item exposure, approximately 1000 ml sludge was withdrawn from activated sludge vessel B and the above culturing procedure was stopped and was used as microbial inoculums in the test, the vessel was identified as B' and aerated at 23.0-25.2°C. On the day of the test, 10 ml of the sludge with five replicates were taken out from vessel B' and weighed and then dried at 105 °C for about 1.5 h.
- Concentration of sludge: concentration of suspended sludge was 4782 mg SS/L, from this result, the added amount of sludge was calculated for 100 mg SS/L
- Water filtered: yes
- Type and size of filter used: fine sieve- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):

Duration of test (contact time):

28 d

Initial conc.:

30.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The stock solutions were prepared as the following usmg analytical grade reagents: (A) 0.8507 g of potassium acid phosphate, 2.1753 g of dipotassium hydrogen phosphate, 4.4609 g of dibasic sodium phosphate dodecahydrate and 0.1704 g of ammonium chloride were dissolved in deionised water and the volume was made up to 100 mL. The pH of the solution was 7.27. (B) 2.2501 g of magnesium sulfate heptahydrate was dissolved in deionised water and the volume was made up to 100 mL. (C) 2.7508 g of calcium chloride was dissolved in deionised water and the volume was made up to 100 ml. (D) 0.0253 g of iron (III) chloride hexahydrate was dissolved in deionised water and the volume was made up to 100 mL. For 1 liter of mineral medium, 3 ml each of solution (A), (B), (C) and (D), and deionised water was made up to 1000 mL. 5 L of mineral medium were prepared for the test.
- pH: The pH of mineral medium was 7.61 - 7.70.
- Test temperature: 20 ± 1°C (actual monitoring temperature 19.61-20.19°C)
- Suspended solids concentration: 100 mg/mL
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Some mineral medium was added to 1 L flask, 6.27 mL of the prepared activated sludge was added to flask and made up to volume to give a suspended solid concentration of 30 mg/L. Some mineral medium was added to 2 L flask, 41.824 mL of the prepared activated sludge was added to flask and made up to volume to give a suspended solid concentration of 100 mg/L. Some mineral medium was added to 1 L flask, 20.912 mL of the prepared activated sludge was added to flask and made up to volume to give a suspended solid concentration of 100 mg/L. 3L in total were prepared.
- Number of culture flasks/concentration: 3

SAMPLING
- Sampling frequency: The BOD values in all test vessels were recorded on a daily basis over the 28 day period by the automated respirometer
- Sampling method: BOD sensors
- Sample storage before analysis: All samples analyzed the same day

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic control: Yes
- Toxicity control: Yes

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

22.3

Sampling time:

28 d

Remarks on result:

other: inherently biodegradable

Key result

Parameter:

% degradation (test mat. analysis)

Value:

80

Sampling time:

28 d

Remarks on result:

other: primary biodegradable

Details on results:

Based on the calculation for BOD, the percentage biodegradation after 28 days averaged 22.3% (triplicate). The test item exhibited a percentage biodegradation exceeding the pass level of 20% after 28 days and demonstrated inherent and primary biodegradability. Based on the residual test item from HPLC analysis after treatment of 28 days, the primary percentage biodegradation averaged 80.0% (triplicate). The percentage degradation of sodium benzoate (reference item) reached 79.5% after the seventh day and 85.3% at the fourteenth day. According to the criterion of OECD and MEP, P.R.China Guideline for the Testing of Chemicals, the test was considered as valid.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Based on the grading criteria in the Guidelines for the Hazard Evaluation of New Chemical Substances, State Environmental Protection Agency of P.R.C. (HJ/T 154-2004), the test item met criteria for "inherent and primary biodegradability" under the described test conditions.

Executive summary:

This test assessed the inherent biodegradability of the test item on micro-organisms in an aerobic aqueous medium by measuring the Biochemical Oxygen Demand (BOD) according to OECD 302B under GLP. The residual amount of the test item in test vessels after 28 days incubation was also determined by HPLC method. The concentration of test item in Test Suspension BOD bottles was at a level of 30.5 mg/L (triplicate), the inoculums concentration was 100 mg/L as suspended solid. Based on the calculation for BOD, the percentage biodegradation after 28 days averaged 22.3% (triplicate). The test item exhibited a percentage biodegradation exceeding the pass level of 20% after 28 days and demonstrated inherent and primary biodegradability. Based on the residual test item from HPLC analysis after treatment of 28 days, the primary percentage biodegradation averaged 80.0% (triplicate). The percentage degradation of sodium benzoate (reference item) reached 79.5% after the seventh day and 85.3% at the fourteenth day. According to the criterion of OECD and MEP, P.R.China Guideline for the Testing of Chemicals, the test was considered as valid. Based on the grading criteria in the Guidelines for the Hazard Evaluation of New Chemical Substances, State Environmental Protection Agency of P.R.C. (HJ/T 154-2004), the test item met criteria for "inherent and primary biodegradability" under the described test conditions

Description of key information

Not readily biodegradable, CO2 Evolution (Modified Sturm) Test, OECD 301B, Hugill 2014

Inherently biodegradable, Biochemical Oxygen Demand, OECD 302C, Xue 2015

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater

Additional information

Ready Biodegradation was assessed using the CO2 Evolution (Modified Sturm) Test according to OECD 301B under GLP. The mean cumulative percentage biodegradation of the test item in the test media at the end of the test (Day 28) was 1.8%. In accordance with the guideline, the test item was therefore not ready biodegradable under the test conditions. In the toxicity control, containing both test item and the reference item, sodium benzoate, the cumulative percentage biodegradation was greater than 35% (45.5%) based on total TOC. According to the guideline, this indicates that the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration. The cumulative percentage biodegradation in the toxicity control was similar to the procedural controls containing sodium benzoate only (90.7%), if calculated only on the amount of sodium benzoate added. In the procedure controls, the reference item sodium benzoate was degraded by an average of 85.0% by exposure Day 12 and reached an average cumulative percentage biodegradation of 91.0% by the end of the test (Day 28), thus confirming suitability of the activated sludge.

Apart from the ready biodregradation, the inherent biodegradability was assessed using the Inherent Biodegradability Test. This test assessed the inherent biodegradability of the test item on micro-organisms in an aerobic aqueous medium by measuring the Biochemical Oxygen Demand (BOD) according to OECD 302B under GLP. And the residual amount of the test item in test vessels after 28 days incubation was also determined by HPLC method. The concentration of test item in Test Suspension BOD bottles was at a level of 30.5 mg/L (triplicate), the inoculums concentration was 100 mg/Las suspended solid. Based on the calculation for BOD, the percentage biodegradation after 28 days averaged 22.3% (triplicate). The test item exhibited a percentage biodegradation exceeding the pass level of 20% after 28 days and demonstrated inherent and primary biodegradability. Based on the residual test item from HPLC analysis after treatment of 28 days, the primary percentage biodegradation averaged 80.0% (triplicate). The percentage degradation of sodium benzoate (reference item) reached 79.5% after the seventh day and 85.3% at the fourteenth day. According to the criterion of OECD and MEP, P.R.China Guideline for the Testing of Chemicals, the test was considered as valid. Based on the grading criteria in the Guidelines for the Hazard Evaluation of New Chemical Substances, State Environmental Protection Agency of P.R.C. (HJ/T 154-2004), the test item met criteria for "inherent and primary biodegradability" under the described test conditions.