tert-butyl {(1S)-2-[(1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hex-2-yl]-1-[3-hydroxyadamantan-1-yl]-2-oxoethyl}carbamate

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:

The study was conducted between 01 February 2012 and 7 March 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Identification: BMS-528235-01
Description: off white powder
Batch: 1L63487N
Purity: 98.6 %
Expiry / retest date: not supplied
Storage conditions: room temperature in the dark
A Certificate of Analysis supplied by the sponsor is given in Appendix 1 attached in overall remarks and attachments section.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on
6 February 2012 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.


Preparation of Inoculum
The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.7 g/L prior to use.
* Rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven


The mineral medium used in this study was that recommended in the OECD Guidelines.
Appendix 2 Mineral Medium
Solution a KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L
NH4Cl 0.50 g/L

pH = 7.4

Solution b CaCl2 27.50 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeCl3.6H2O 0.25 g/L

To 1 liter (final volume) of purified water* was added the following volumes of solutions
a – d.

10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

15 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preliminary Investigational Work
In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, samples are taken for Dissolved Organic Carbon (DOC) analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analyzed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-VCPH TOC analyzer (see Appendix 3 in any other information on materials and methods including tables section).

An amount of test item (100 mg) was dissolved in mineral medium (1 liter) to give a 100 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further amount of test item (100 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 liter. Two samples were taken for DOC analysis; one after filtration through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as per the test item samples.

Experimental Preparation
For the purpose of the test, the test item was dissolved directly in mineral medium.

An amount of test item (300 mg) was dissolved in mineral medium with the aid of ultrasonication (45 minutes) and the volume adjusted to 1 liter to give a 300 mg/L stock solution. An aliquot (150 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final concentration of 15.0 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the test item was inverted several times to ensure homogeneity of the solution.

A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Data from the inoculum control vessels was shared with similar concurrent studies.

Reference Item
For the purposes of the test, a reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 minutes. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Data from the procedure control vessels was shared with similar concurrent studies.


Toxicity Control
For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

An aliquot (150 mL) of the test item stock solution was dispersed in inoculated mineral medium along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was adjusted to 3 liters to give a final concentration of 15.0 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.


Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:

a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final conc entration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at approximately 21 DegC, in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 33.3 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. If necessary the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.


Sampling and Analysis
CO2 Analysis
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29.

The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analyzed for CO2 immediately.

On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

The samples were analyzed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyzer. Samples (300 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyzer. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Inorganic Carbon/Total Carbon Analysis
Samples (30 mL) were removed from the inoculum control and test item vessels on Day 0 and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis.

The samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer. Samples (50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyzer. Total carbon analysis is carried out at 680 DegC using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in triplicate.


pH Measurements
The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter

Evaluation of Data
Calculation of Carbon Content
The theoretical amount of carbon present in the test item for BMS-528235-01 (C23H33N3O4) was calculated as follows:#

(No of carbon atoms x molecular weight of carbon/molecular weight of test item) x 100

[(23 X 12.011)/415.53] x 100 = 66.48%

Thus for a concentration of 10 mg C/L (a total of 45.0 mg of test item in 3 liters) the total organic carbon present was 30 mg C.

The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated as follows:

(No of carbon atoms x molecular weight of carbon/molecular weight of sodium benzoate) x 100

[(7 X 12.011)/144.11] x 100 = 58.34%

Thus for a 10 mg C/L test concentration (a total of 51.4 mg of sodium benzoate in 3 liters) the total organic carbon present for sodium benzoate was 30 mg C.


Percentage Degradation
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1 in any other information on materials and methods including tables section, into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference items before substitution in the equation.

%ThCo2(=%degradation*)=mgICin test flask-mgICin control divided by mg TOC added as test chemical multiplied by 100%.

* The conversion factor for carbon to carbon dioxide is 3.67

The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total Co2 evolution = mgIC in control x 100/%C of Co2 x 1/test volume.

Validation criteria:
The results of the degradation test are considered valid if in the same test the reference item yields equal to or greater than 60% degradation by Day 14.

The test item may be considered to be readily biodegradable if equal to or greater than 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.

The toxicity control (test item and sodium benzoate) should attain equalt to or greater than 25% degradation by Day 14 for the test item to be considered as non-inhibitory.

The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the time the plateau is reached, at the end of the test or at the end of the 10-Day window, as appropriate, is less than 20%.

The total CO2 evolution in the inoculum control vessels at the end of the test should not normally exceed 40 mg/L medium (= 120 mg/3 liters, corresponding to 33 mg C per flask), however values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.

The IC content of the test item suspension in the mineral medium at the beginning of the test should be <5% of the TC.

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

Preliminary study:

The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge (see Appendix 3 in any other information on materials and methods including tables section ). Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.

Test performance:

The total CO2 evolution in the inoculum control vessels on Day 28 was 34.35 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1 attached in overall remarks and attachements section. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on Days 0 and 28 are given in Table 4. Observations made on the contents of the test vessels are given in Table 5.
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

All tables are in any other information on materials and methods including tables section.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test item attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Although the toxicity control vessel obtained greater than 25% degradation by Day 14 of the study, indicating the test item to be not classed as exhibiting any inhibitor effects to the activated sewage sludge, between Days 14 and 28 the degradation value for the toxicity control vessel decreased from 28% degradation to 23% degradation.

It is also clear from the test item results that some inhibition of the activated sewage sludge micro-organisms had occurred as the CO2 evolution rates in the test item vessels were significantly lower than those in the control vessel after 28 days.

Care should therefore be taken in the interpretation of the results due to the inhibitory nature of the test item to the activated sewage sludge micro-organisms used in the study.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 63% degradation after 14 days and 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Table1     Inorganic Carbon Values on Each Analysis Occasion

Day	Inoculum Control (mg IC)				Sodium Benzoate Procedure Control (mg IC)				Test Item (mg IC)				Test Item plus Sodium Benzoate Toxicity Control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	2.57	1.52	1.98	1.52	1.87	1.52	1.75	1.40	1.87	1.52	1.63	2.45	1.75	1.63
2	2.67	-	6.61	-	20.53	-	18.21	-	5.11	-	5.80	-	18.91	-
6	12.69	-	13.15	-	29.06	-	27.79	-	11.19	-	12.46	-	28.72	-
8	18.46	-	17.43	-	35.66	-	34.29	-	15.59	-	17.31	-	36.35	-
10	18.13	-	17.55	-	41.15	-	38.65	-	16.30	-	17.67	-	36.71	-
14	20.74	-	20.63	-	40.23	-	39.21	-	17.57	-	18.59	-	37.29	-
21	23.66	-	25.24	-	46.98	-	43.71	-	22.08	-	22.65	-	41.57	-
28	27.22	-	29.01	-	45.02	-	48.16	-	23.41	-	23.63	-	43.91	-
29	30.95	3.48	30.62	2.78	49.32	2.90	50.21	2.90	24.61	3.13	24.94	3.25	44.69	44.69

R₁– R₂          =   Replicates 1 and 2

Abs              =  CO₂absorber vessels

Table 2     Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate Procedure Control	% Degradation Test Item	% Degradation Test Item plus Sodium Benzoate Toxicity Control
0	0	0	0
2	49	3	24
6	52	0	26
8	57	0	31
10	74	0	31
14	63	0	28
21	70	0	29
28	62	0	26
29*	63	0	23

*Day 29 values corrected to include any carry-over of CO₂detected in Absorber 2

Table 3     Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon* (mg/L)	Inorganic Carbon* (mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	8.63	-0.17	0
Test Item 10 mg C/L R2	9.02	0.34	4

R₁– R₂     =  Replicates 1 and 2

*       Corrected for control values. Negative values are due toasured concentrations being less than control values

Table 4     pH Values of the Test Preparations on Days 0 and 28

Test Vessel	pH prior to adjustment on Day 0	pH after adjustment on Day 0	pH on Day 28
Inoculum ControlR1	7.6	-	7.6
Inoculum Control R2	7.6	-	7.5
Procedure Control 10 mg C/L R1	7.7	7.6	7.6
Procedure Control 10 mg C/L R2	7.7	7.6	7.6
Test Item 10 mg C/L R1	7.7	7.6	7.5
Test Item 10 mg C/L R2	7.6	-	7.5
Toxicity Control 20 mg C/L	7.7	7.6	7.6

 R₁– R₂    =      Replicates 1 and 2

-                          =     No adjustment necessary

Table 5     Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 13	Day 20	Day 27
Inoculum Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Procedure Control	R1	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
	R2	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
Test Item	R1	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface
	R2	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test item visible. Small amount of foam on surface
Toxicity Control		Light brown dispersion, no undissolved test or reference item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test or reference item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test or reference item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test or reference item visible. Small amount of foam on surface	Light brown dispersion, no undissolved test or reference item visible. Small amount of foam on surface

R₁– R₂= Replicates 1 and 2

Appendix 3   Dissolved Organic Carbon (DOC) Values from the Preliminary Investigational Work

In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge the following samples were analysed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-V_CPHTOC analyzer.

 

Sample	DOC Concentration		% of Nominal Carbon Content
	mg C/L	mg C/L corrected for appropriate control
Mineral medium	<LOQ	-	-
Control, inoculated at 30 mg ss/L, Filtered	<LOQ	-	-
Control, inoculated at 30 mg ss/L, Centrifuged	<LOQ	-	-
100 mg/L Untreated	64.26	64.26	97
100 mg/L Filtered	64.29	64.29	97
100 mg/L, inoculated at 30 mg ss/L, Filtered	66.15	66.15	100
100 mg/L, inoculated at 30 mg ss/L, Centrifuged	66.47	66.47	100

 

These results indicated that the test item did not adsorb to filter matrices or activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without causing a loss of any test item.LOQ

 

LOQ= Limit of Quantitation (determined down to 1.0 mg C/L).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Care should be taken in the interpretation of the results due to the inhibitory nature of the test item to the activated sewage sludge micro-organisms used in the study.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Care should be taken in the interpretation of the results due to the inhibitory nature of the test item to the activated sewage sludge micro-organisms used in the study.

Although the toxicity control vessel obtained greater than 25% degradation by Day 14 of the study, indicating the test item to be not classed as exhibiting any inhibitor effects to the activated sewage sludge, between Days 14 and 28 the degradation value for the toxicity control vessel decreased from 28% degradation to 23% degradation.

It is also clear from the test item results that some inhibition of the activated sewage sludge micro-organisms had occurred as the CO2 evolution rates in the test item vessels were significantly lower than those in the control vessel after 28 days.

Executive summary:

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO₂Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

 

Methods

The test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21 °C for 28 days.

 

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes

 

Results and Conclusion

The test item attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

 

Although the toxicity control vessel obtained greater than 25% degradation by Day 14 of the study, indicating the test item to be not classed as exhibiting any inhibitory effects to the activated sewage sludge, between Days 14 and 28 the degradation value for the toxicity control vessel decreased from 28% degradation to 23% degradation.

 

It is also clear from the test item results that some inhibition of the activated sewage sludge micro-organisms had occurred as the CO₂evolution rates in the test item vessels were significantly lower than those in the control vessel after 28 days.

 

Care should therefore be taken in the interpretation of the results due to the inhibitory nature of the test item to the activated sewage sludge micro-organisms used in the study.