N-(dodecylphenyl)naphthalen-1-amine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 May 1999 to 23 June 1999.

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)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Further details not specified in the study report.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 24 May 1999 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.

Duration of test (contact time):

28 d

Initial conc.:

17.1 mg/L

Based on:

test mat.

Remarks:

equivalent to 10 mg C/l

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Experimental Preparation
Following the recommendations of the International Standards Organisation, for the purpose of the definitive study the test material was prepared by adsorption onto silica gel prior to dispersion in culture medium in order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.
An amount of test material (34.5 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230 - 400 mesh Sigma Lot No 721-1351 5) prior to dispersal in approximately 100 ml of culture medium with the aid of high shear mixing (7500 rpm, 10 minutes). The test material/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 11 -5 mg/l, equivalent to 10 mg carbon/l.

Standard Material
For the purposes of the study a standard material, sodium benzoate (C6H5COONa) (Sigma Lot No 125H0060), was used. An initial stock solution of 1000 mg/l was prepared by direct solution in culture medium and a 51.4 ml aliquot added to the test vessel to give a final test concentration of 17.1 mg/l, equivalent to 10 mg carbon/l.
Silica gel (100 mg) was also added to the standard vessel in order to maintain consistency between the test and standard material vessels.

METHODS
Procedure
Preparation of inoculum
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. A subsample of the washed sewage sludge was then removed and the suspended solids concentration determined.

Culture medium
The culture medium used in this study was that recommended in the OECD Guidelines.

Preparation of test system
The following test solutions were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium plus 100 mg of silica gel.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium plus 100 mg sf silica gel to give a final concentration of 10 mg carbon/l.
C) The test material, in duplicate, in inoculated culture medium plus 100 mg of silica gel to give a final concentration of 10 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium plus 100 mg of silica gel to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).

Silica gel was added to the control and standard material vessels in order to maintain consistency between these vessels and the test material vessels.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The study was carried out in a temperature controlled room at 21 °C, in darkness.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 30 ml of inoculum and aerated overnight. On day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by sparging 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 vessel on days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on days 0 and 29.
The samples taken on days 0 Absorber 1, 1, 2, 3, 8, 10, 14, 16, 20, 22, 24, 27 and 28 were analysed for CO2 immediately. The test material replicates R1 and R2 Day 2 and Day 3 samples were re-analysed due to erroneous values obtained during the original analyses. The samples were stored deep frozen at -20 °C prior to re-analysis. The samples taken on Day 0 from Absorber 2 and on Day
29 from Absorbers 1 and 2 were stored deep frozen at -20 °C prior to analysis. The samples taken on days 6, 12 and 18 were also stored deep frozen at -20 °C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.
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 analysed for CO2 using an lonics 1555B TOC analyser and a Dohrmann DC-190 TOC analyser. Samples (40 or 50 μl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample by phosphoric acid using nitrogen (oxygen free) or research grade oxygen as the carrier gas.
Calibration was by a standard solution of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
On days 0 and 28 samples (20 ml) were removed from the control and standard vessels and filtered through Gelman 0.45 μm Acrocap filters (approximately 5 ml discarded) prior to Dissolved Organic Carbon (DOC) analysis.
DOC analysis of the test material dispersions was not possible due to the insoluble nature of the test material in water.
Samples (20 ml) were removed from the test and toxicity control culture vessels on day 0 prior to the addition of the test material in order to calculate the TC/IC ratio in the test media. The samples were filtered through Gelman 0.45 μm Acrocap filters (approximately 5 ml discarded) prior to Dissolved Organic Carbon (DOC) analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (26, 22 or 13 μl) were injected into the Total Carbon (TC) and inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680 °C using a platinum catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

Toxicity determination
A toxicity control (APAN and sodium benzoate) was included in the study to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the study.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not specified

Test performance:

The total CO2 evolution in the control vessels on day 28 was 36.55 mg/l (= 109.65 mg/3 I) and therefore satisfied the validation criterion given in the OECD Guidelines.
The TC/IC ratio in the test vessels on day 0 did not exceed 5% and therefore satisfied the test guidelines.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

28 d

Details on results:

The test material attained 3% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.
The results of the inorganic carbon analysis of samples from the first absorber vessels on day 29 showed an increase in all replicate vessels. These increases are considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on day 28 and resulted in a decrease in the percentage degradation value for the test material from 3% on day 28 to 2% on day 29. This is considered to be due to the increases in inorganic carbon within the replicate control vessels being greater than those within the replicate test 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 toxicity control attained 29% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study. The increase in inorganic carbon in the first absorber vessels on day 29 resulted in an increase in the percentage degradation value for the toxicity control from 29% on day 28 to 34% on day 29.
The increase in inorganic carbon in the first absorber vessels on day 29 resulted in an increase in the percentage degradation value for the standard material from 81 % on day 28 to 84% on day 29.
Analysis of standard material culture vessels on days 0 and 28 for dissolved organic carbon (DOC) gave a percentage degradation value of 92%.

Results with reference substance:

Sodium benzoate attained 81% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

INORGANIC CARBON VALUES ON EACH ANALYSIS OCCASION

DAY	Control (mg IC)				Sodium benzoate (mg IC)				APAN (mg IC)				APAN plus sodium benzoate toxicity control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs 1	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	1.87	1.05	1.05	0.70	1.05	1.05	0.70	0.82	1.05	0.82	0.82	0.93	1.63	0.82
1	1.74	-	2.44	-	2.78	-	2.90	-	1.97	-	3.25	-	4.52	-
2	5.54	-	4.15	-	8.07	-	12.11	-	5.54	-	5.19	-	11.65	-
3	5.96	-	5.73	-	13.76	-	15.94	-	5.85	-	5,96	-	12.15	-
8	14.95	-	11.90	-	32.30	-	37.17	-	11.56	-	14.28	-	27.43	-
10	14.87	-	12.62	-	34.03	-	37.74	-	13.63	-	14.98	-	28.84	-
14	15.47	-	15.59	-	37.52	-	39.30	-	15.36	-	17.03	-	31.40	-
16	15.83	-	16.38	-	38.95	-	40.50	-	16.49	-	17.04	-	33.31	-
20	19.46	-	19.13	-	42.31	-	44.06	-	20.12	-	19.90	-	36.41	-
22	24.02	-	22.06	-	45.97	-	48.36	-	23.25	-	24.12	-	41.29	-
24	26.89	-	24.52	-	49.25	-	50.33	-	26.57	-	26.89	-	43.31	-
27	29.41	-	27.37	-	50.45	-	53.77	-	29.41	-	28.87	-	46.26	-
28	30.40	-	29.44	-	52.48	-	56.21	-	31.25	-	30.61	-	47.15	-
29	31.27	2.55	31.06	2.32	55.44	2.90	56.50	2.90	32.22	2.09	31.38	2.32	50.35	3.37

R₁– R₂= Replicates               Abs = CO₂absorber vessel     “-“ = value not determined

 

PERCENTAGE BIODEGRADATION VALUES

Day	% Degradation Sodium benzoate	% Degradation APAN	% Degradation APAN plus sodium benzoate toxicity control
0	0	0	0
1	3	2	4
2	17	2	11
3	30	0	11
8	73	0	24
10	74	2	25
14	76	2	26
16	79	2	29
20	80	2	29
22	80	2	30
24	80	3	29
27	79	3	30
28	81	3	29
29*	84	2	34

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

 

TOTAL AND INORGANIC CARBON VALUES IN THE CULTURE VESSELS ON DAY 0

Test vessel	Total Carbon corrected for control (mg/l)	Inorganic Carbon corrected for control (mg/l)	% Inorganic Carbon
Sodium benzoate 10 mg C/l R1	10.28	-0.08	-1*
Sodium benzoate 10 mg C/l R2	9.69	0.33	3
APAN 10 mg C/l R1	0.84**	0.17	2
APAN 10 mg C/l R2	9.87**	0.13	1
APAN plus sodium benzoate Toxicity control 20 mg C/l	19.59**	0.14	1

R₁– R₂= Replicate 1 and 2

*Negative values are considered to be due to sampling and/or analytical variation between control and test vessels.

**Result given is the sum of the results of TC analysis plus the nominal TC contribution of test or test/standard materials.

 

DISSOLVED ORGANIC CARBON (DOC) VALUES IN THE CULTURE VESSELS ON DAYS 0 AND 28

DOC* Concentration in the culture vessel
Test vessel	Day 0		Day 28
	mg C/l	% Nominal carbon content	mg C/l	% of initial carbon concentration	% Degradation
Sodium benzoate 10 mg C/l R1	10.37	104	0.81	8	92
Sodium benzoate 10 mg C/l R2	9.36	94	0.77	8	92

R₁– R₂= Replicates 1 and 2

*Corrected for control values

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test material attained 3% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.

Executive summary:

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

 

The test material was exposed to activated sewage sludge micro-organisms at a concentration of 10 mg C/I with culture medium in sealed culture vessels in the dark at 21 °C for 28 days. The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

 

Following the recommendations of the international Standards Organisation, the test material was adsorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

 

The test material attained 3% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.

Description of key information

GLP accredited laboratory study was performed to assess the ready biodegradability of the test material using the CO2 Evolution Test in accordance with OECD Guideline 301B, EU Method C.4 -C and US EPA OPPTS 835.3110.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media.

 

The test material was exposed to activated sewage sludge micro-organisms at a concentration of 10 mg C/I with culture medium in sealed culture vessels in the dark at 21 °C for 28 days. The degradation of the test material was assessed by the determination of carbon dioxide produced.

 

The test material attained 3% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.