Tricyclo[3.3.1.13,7]decane-1-acetic acid, .alpha.-[[(1,1-dimethylethoxy)carbonyl]amino]-3-hydroxy-, (.alpha.S)-

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Decmber 2004 to 27 April 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted in 2005 according to OECD Method # 301B and EU method C.4-C and in accordance with GLP. The study material is well characterized.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-{[(tert-butoxy)carbonyl]amino}-2-(3-hydroxyadamantan-1-yl)acetic acid

EC Number:

700-361-0

Cas Number:

361442-00-4

Molecular formula:

C17H27NO5

IUPAC Name:

(2S)-2-{[(tert-butoxy)carbonyl]amino}-2-(3-hydroxyadamantan-1-yl)acetic acid

Constituent 2

Reference substance name:

1-Hydroxyadamantanyl-3-(S)-Boc-glycine

IUPAC Name:

1-Hydroxyadamantanyl-3-(S)-Boc-glycine

Details on test material:

White powder; stored at room temperatur; received at testing laboratory on 06 October 2004. Based on the test material empirical formula, its carbon content is 62.8 w/w% .

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None used.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A sample of activated sludge was collected from one of the return lines of the Burley Menston sewage treatement works located in West Yorkshire, UK. Upon receipt the sample of activated sludge is maintained on continuous aeration.

Duration of test (contact time):

0 - 28 d

Initial test substance concentration

Initial conc.:

15 other: mgC/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

The test was conducted in an aqueous synthetic mineral salts medium. On the day before the start of the test, a test medium concentrate was prepared containing 30 mL/L of solution (a) and 3 mL/L of each of solutions (b), (c) and (d). Solutions (a) to (d) were nominally composed as follows:
(a) 8.50 g potassium dihydrogen phosphate (BDH); 21.75 g dipotassium hydrogen phosphate (BDH); 33.40 g disodium hydrogen phosphate dihydrate (Fisher); 0.50 g ammonium chloride (Sigma), all dissolved in and made up to 1 L with reverse-osmosis water;
(b) 36.40 g calcium chloride dihydrate (BDH), dissolved in and made up to 1 L with reverse-osmosis water;
(c) 22.50 g magnesium sulphate heptahydrate (BDH), dissolved in and made up to 1 L with reverse-osmosis water;
(d) 0.25 g ferric chloride hexahydrate (Aldrich) plus one drop concentrated hydrochloric acid (BDH), dissolved in and made up to 1 L with reverse-osmosis water.

On the basis of the suspended solids determination described above, the medium was inoculated with activated sludge at 90 mg suspended solids/L to provide a nominal final solids concentration of 30 mg/L in each test vessel. A 1 L aliquot of the inoculated medium concentrate was added to each vessel, followed by 1.5 L of ultrapure water. The vessels were then sealed and aerated overnight with CO2-free air. Dosing of the test and reference substances was carried out the following day.

Dosing the Test Substance
The test substance was insufficiently soluble to allow the preparation of an aqueous stock solution. Appropriate weighings (nominally 71.8 mg) of the test substance were made into 50 mL volumetric flasks that were made up to the mark with ultrapure water and treated with ultrasound for 30 minutes. The contents of each flask were then added to the appropriate test vessel. Each flask was then made up to the mark with ultrapure water and this rinse added to the respective test vessel. This rinse procedure was repeated three more times to give a total addition of 250 mL. Additions were made, as described to the two test vessels and to the toxicity control to give a final nominal test concentration of 15 mgC/L.

Dosing the Reference Substance
A stock solution at 2.25 gC/L was prepared by dissolving approximately 3.859 g sodium benzoate in 1000 mL reverse-osmosis water. Reference and toxicity control vessels were dosed by addition of 20 mL of the stock solution, to give a nominal sodium benzoate concentration corresponding to 15 mgC/L.

Once all additions were complete, the volume in each vessel was raised to 3 L by addition of ultrapure water. Each vessel was then sealed, connected to a series of three traps containing aqueous barium hydroxide, and the air supply restored at a flow rate of approximately 50 mL per minute.

The test material at a concentration of 15 mg carbon per liter was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at 22 +- 2 C for 28 days. The degradation of the test material was assessed by determination of carbon dioxide produced.

On Day 28 of the test, each culture vessel was opened and 1 mL of concentrated hydrochloric acid added. The vessels were then reconnected to the train of trap bottles and aeration continued until the following day. The acidification and aeration procedure drives off generated carbon dioxide remaining in solution. Final sampling and titrations were carried out on Day 29 when all of the traps in each train were sampled. Titration results from traps removed on Day 29 are reported as Day 28 as the acidification halts biological activity, hence preventing further biodegradation after Day 28.

Reference substance

Reference substance:

other: sodium benzoate

Results and discussion

Preliminary study:

Not completed.

Test performance:

All the validity criteria were met and the results of this study may therefore be considered valid.

The following validity criteria stated in the protocol were satisfied:
-The IC content of the test medium was satisfactory at the start of the study, corresponding to less than 5% of the total carbon based on the nominal dose level (initial IC content was 1.3% of the nominal dose level).
- The percentage degradation of the reference substance, sodium benzoate, reached 60% of the theoretical maximum within 14 days (surpassed on Day 8).
- the individual carbon dioxide evolution data recorded in duplicate test vessels lay within 20% of one another at the end of the test.
- the final cumulative CO2 yield from the blank vessels did not exceed 120 mg per three litres of medium (total mean blank CO2 production was 99.4 mg per 3 L).

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

2

Sampling time:

1 d

Remarks on result:

other: mean

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

2

Sampling time:

8 d

Remarks on result:

other: mean

% Degradation 3

Parameter:

% degradation (CO2 evolution)

Value:

2

Sampling time:

15 d

Remarks on result:

other: mean

% Degradation 4

Parameter:

% degradation (CO2 evolution)

Value:

2

Sampling time:

22 d

Remarks on result:

other: mean

% Degradation 5

Parameter:

% degradation (CO2 evolution)

Value:

2

Sampling time:

28 d

Remarks on result:

other: mean

Details on results:

BMS-528233-01 did not show evidence of significant biodegradation during the test and achieved a mean value of 2% on Day 28. BMS-528233-01 cannot therefore, be considered to be readily biodegradable.
Final CO2 yields for the test substance, expressed as a percentage of theoretical, were 0% in replicate 1 and 4% in replicate 2. Maximum divergence between replicates was 4%.
The measured pH was 7.3 in all vessels measured on Day 0 and ranged from 7.5 to 7.6 on Day 28.
The IC concentration of the test inoculated mineral salts medium was found to be 0.5744 mgC/L. After dilution, this corresponds to an IC content of 0.1915 mgC/L in the test cultures, equivalent to 1.3% of the carbon loading of 15 mgC/L.
Assessment of degradation in the toxicity control was confined to the sodium benzoate fraction. Toxicity controls: The rate of CO2 production generally kept pace with that observed in the two reference vessels containing sodium benzoate alone. The level of degradation was 64% on Day 8 and was 83% at the end of the test. The absence of any suppression with respect to the performance of the sodium benzoate alone demonstrates that the BMS-528233-01 present in the toxicity control did not inhibit the microbial degradation of the reference substance.

BOD5 / COD results

Results with reference substance:

Sodium Benzoate Rapid CO2 generation commenced immediately, slowing to a more gradual rate around Day 3 and again on Day 11,.Both replicates had exceeded 60% biodegradation by Day 8, and the mean level of degradation on Day 28 was 85%. Maximum divergence between the replicates was 2%.

Any other information on results incl. tables

Theoretical CO2 Yields: Cumulative CO2 production data are presented in Table 2. The theoretical yield for the quantity of BMS-528233-01 applied in the test vessels and toxicity control vessel was 165 mg CO2. Theoretical yields for the quantities of applied sodium benzoate in the toxicity control vessel and reference vessels were also 165 mg CO2. The combined theoretical yield from the toxicity control vessel was 330 mg CO2. However, the purpose of this control was not to assess the extent of degradation of the entire mixture, but instead to assess the impact of the presence of the test substance on the degradation of the reference material. The theoretical yield in this case was, therefore, limited to the 165 mg CO2expected from the sodium benzoate alone.

Table 2

Evolved CO2(mg)
Day 1		Day 2	Day 3	Day 6	Day 8	Day 11	Day 15	Day 18	Day 22	Day 25	Day 28
Mean Control	13.3	22.5	40.2	58.6	69.2	77.6	84.5	88.6	91.8	94.1	99.4
Test: Replicate 1	13.0	22.1	39.7	58.6	68.8	77.3	84.3	88.7	92.4	94.6	100.1
Test: Replicate 2	19.7	29.8	47.3	65.1	75.2	83.1	90.3	94.6	98.0	100.5	106.1
Reference: Replicate 1	47.2	88.7	122.6	156.6	176.2	192.9	207.1	216.0	223.5	228.9	238.8
Reference: Replicate 2	44.2	85.4	119.0	154.8	175.8	193.7	209.1	218.5	226.1	230.9	239.9
Toxicity control	44.1	85.6	119.7	154.5	174.6	191.3	205.8	214.9	223.3	228.2	236.8

Measured CO2Yields as a Percentage of Theoretical Biodegradation (Dt) of the reference substance and of BMS-528233-01 expressed in terms of percentage theoretical CO 2yield was calculated by applying the formula:

Dt = (cumulative mg CO₂produced at time t /165) x 100

Percentage Biodegradation
(%)
Day		Day	Day	Day	Day	Day	Day	Day	Day	Day	Day
1		2	3	6	8	11	15	18	22	25	28
Test: Replicate 1	0	0	0	0	0	0	0	0	0	0	0
Test: Replicate 2	4	4	4	4	4	3	4	4	4	4	4
Mean	2	2	2	2	2	2	2	2	2	2	2
Reference: Replicate 1	21	40	50	59	65	70	74	77	80	82	84
Reference: Replicate 2	19	38	48	58	65	70	75	79	81	83	85
Mean	20	39	49	59	65	70	75	78	81	82	85
Toxicity control	19	38	48	58	64	69	74	77	80	81	83

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Mean carbon dioxide evolution from BMS-528233-01 reached 2% of the theoretical maximum at the applied concentration over the course of the 28 day incubation. The level of biodegradation failed to meet the requirements for ready biodegradability and BMS-528233-01 cannot, therefore, be classified as readily biodegradable.

Executive summary:

The ready biodegradability of BMS-528233-01 was assessed by measurement of carbon dioxide evolution under standard conditions. The procedure followed was method C.4-C described in Annex V of EU Directive 67/548/EEC. The method is identical to the 1992 revision of OECD Guideline 301B and that in section (m) of US EPA OPPTS method 835.3110.

Degradation of the reference substance, sodium benzoate, exceeded 60% in both replicates by Day 8 and achieved a mean value of 85% at the end of the test. These data show that the inoculum was viable and exerting normal degradative activity. In the toxicity control group, degradation of sodium benzoate was essentially unaffected and was 64% and 83% on Days 8 and 28, respectively. Mean CO2production in the control cultures was 99.4 mg/3 litres at the end of the test. All validity criteria pertaining to this test type were satisfied, the results are therefore, considered to be valid.

Mean carbon dioxide evolution from BMS-528233-01 was 2% of the theoretical maximum at the applied concentration on Day 28.BMS-528233-01 cannot, therefore, be considered to be readily biodegradable.

The ready biodegradability methods were devised, not as simulations of real aquatic environments, but as stringent fail-safe tests to screen for and to classify substances that would degrade rapidly and completely in natural water bodies. Consequently, although BMS-528233-01 has failed to qualify for classification as readily biodegradable under the conditions employed in this study, the results of this study do not necessarily suggest that it would persist or accumulate in the environment.