9-Anthracenecarboxylic acid, (triethoxysilyl)methyl ester

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:

27-01-2003 till 07-02-2003

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:

1992-07-17

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 1993

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2003-03-10

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the freezer under nitrogen and protected from light.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspension

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of activated sludge: Freshly obtained from the municipal sewage treatment plant Waterschap de Maaskant, ‘s-Hertogenbosch, Netherlands
- Storage conditions: Under continuous aeration
- Pretreatment and preparation of inoculum for exposure: Before use, the sludge was allowed to settle for 30-90 min. Afterwards the liquid was decanted for use as inoculum at the amount of 10 ml/L of mineral medium.
- Concentration of suspended solids in the concentrated sludge: 3.1 g/L
- Water filtered: Yes
- Type and size of filter used, if any: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q; Millipore Corp., Bedford, Mass., USA).

Duration of test (contact time):

28 d

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 L mineral medium contains: 10 mL of stock solution (A), 1 mL of solutions (B to D) and Milli-RO water. For details of the stock solutions see table 1.
- Test temperature: 22.0 – 23.0°C
- pH: At the start of the test 7.5 – 7.6 and on day 28 7.5 – 8.1 (see table 2).
- pH adjusted: No
- Suspended solids concentration: 3.1 g/L

TEST SYSTEM
- Test vessels: 2 L all-glass brown coloured bottles
- Pre-incubation step of the medium: Mineral components, Milli-RO (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with CO2-free air overnight to purge the system of CO2.
- CO2-free air preparation: A mixture of oxygen (21%) and nitrogen (79%) was led through a bottle, containing 0.5 – 1 L 0.0125 M barium hydroxide (Ba(OH)2) solution to trap CO2, which might be present in small amounts. The CO2-free air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per sec (ca. 30 – 100 mL/min).
- Number of culture flasks: 2 bottles containing the test substance and the inoculum (test suspension), 2 bottles containing only the inoculum (inoculum blank), 1 bottle containing 40 mg/L of the reference substance sodium acetate and the inoculum (positive control) and 1 bottle containing the test substance, reference substance and the inoculum (toxicity control).
- Test method: The test substance (18 mg/L) and reference substance (40 mg/L) were added to the bottles. The volumes of suspensions were made up to 2 L with Milli-RO water. During the test the solutions were continuously stirred.
- Details of trap for CO2: Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. The CO2 produced in each test bottle reacted with the Ba(OH)2 in the gas scrubbing bottle and precipitated out as barium carbonate (BaCO3). The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 standardized HCL.
- CO2 determination method: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. On the 28th day 1 mL of concentrated HCL was added to each bottle. The bottles were aerated overnight to drive of the CO2 present in the test suspension. The final titration was made on day 29.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

9

St. dev.:

2.5

Sampling time:

28 d

Details on results:

The test substance was not soluble in water at a concentration of 1 g/L.The relative degradation values calculated from the measurements performed during the test period revealed 7 – 12% degradation (mean 9%, SD 2.5). The total CO2 release in the blank at the end of the test was 33.8 mg CO2 per 2 L of medium. The results are summarized in tables 3 – 6.

Results with reference substance:

The positive control substance was degraded by 84% within 14 days and ≥ 100 within 23 days. In the toxicity control a degradation of 34% was observed within 14 days. The results are summarized in tables 7 & 8.

Table 3: CO₂ production and percentage biodegradation of the test substance (bottle A)

Day	HCL (0.05 N) titrated mL			Produced CO2(mL HCL)	Produced CO2(mg)	Cumulative CO2(mg)	Degradation* (%)
	Blank (mean)		Bottle A
0	-	-		-	-	-	0
2	43.51	43.38		0.13	0.1	0.1	0
5	43.81	43.33		0.48	0.5	0.7	1
7	44.67	44.61		0.06	0.1	0.7	1
9	45.81	45.24		0.57	0.6	1.4	2
14	44.14	43.15		0.99	1.1	2.4	3
19	45.34	44.22		1.12	1.2	3.7	4
23	46.14	43.93		2.21	2.4	6.1	7
27	39.26	37.80		1.46	1.6	7.7	9
29	47.51	46.58		0.93	1.0	8.7	10
29	48.82	48.25		0.56	0.6	9.3	11
29	48.70	48.00		0.70	0.8	10.1	12

*: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test substance: 87.2 mg CO₂/2 L

Table 4: CO₂ production and percentage biodegradation of the test substance (bottle B)

Day	HCL (0.05 N) titrated mL		Produced CO2(mL HCL)	Produced CO2(mg)	Cumulative CO2(mg)	Degradation* (%)
	Blank (mean)	Bottle B
0	-	-	-	-	-	0
2	43.51	42.62	0.89	1.0	1.0	1
5	43.81	42.64	1.17	1.3	2.3	3
7	44.67	43.90	0.77	0.8	3.1	4
9	45.81	45.56	0.25	0.3	3.4	4
14	44.14	43.46	0.68	0.7	4.1	5
19	45.34	45.26	0.08	0.1	4.2	5
23	46.14	45.13	1.01	1.1	5.3	6
27	39.26	43.82	0.00	0.0	5.3	6
29	47.51	47.92	0.00	0.0	5.3	6
29	48.82	48.06	0.75	0.8	6.2	7
29	48.70	49.11	0.00	0.0	6.2	7

*: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test substance: 87.5 mg CO₂/2 L

Table 5: Comparison of biodegradation of the test substance in bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	∆A-B*
0	0	0	0	0
2	0	1	1	1
5	1	3	2	2
7	1	4	2	3
9	2	4	3	2
14	3	5	4	2
19	4	5	5	1
23	7	6	7	1
27	9	6	7	3
29	10	6	8	4
29	11	7	9	4
29	12	7	9	5

*: Absolute difference in biodegradation between bottles A and B

Table 6: CO₂ production in the blank

Day	HCL (0.05 N) titrated mL		Produced CO2(mL HCL)	Produced CO2(mg)	Cumulative CO2(mg)
	Ba(OH)2*	Blank (mean)
0	-	-	-	-	-
2	47.78	43.51	4.28	4.7	4.7
5	47.97	43.81	4.16	4.6	9.3
7	46.22	44.67	1.55	1.7	11.0
9	48.68	45.81	2.86	3.2	14.1
14	47.60	44.14	3.46	3.8	17.9
19	48.15	45.34	2.81	3.1	21.0
23	49.94	46.14	3.80	4.2	25.2
27	42.28	39.26	3.03	3.3	28.5
29	50.30	47.51	2.79	3.1	31.6
29	50.34	48.82	1.53	1.7	33.3
29	49.15	48.70	0.44	0.5	33.8

*: “Strength” of untreated 0.0125 Ba(OH)₂solution

Table 7: CO₂ production and percentage biodegradation of the positive control substance

Day	HCL (0.05 N) titrated (mL)		Produced CO2(mL HCL)	Produced CO2(mg)	Cumulative CO2(mg)	Degradation* (%)
	Blank (mean)	Positive control
0	-	-	-	-	-	-
2	43.51	34.34	9.17	10.1	10.1	12
5	43.81	23.95	19.86	21.8	31.9	37
7	44.67	29.02	15.65	17.2	49.1	57
9	45.81	36.25	9.56	10.5	59.1	70
14	44.14	32.93	11.21	12.3	72.0	84
19	45.34	37.75	7.59	8.3	80.3	94
23	46.14	41.76	4.38	4.8	85.2	≥100
27	39.26	35.56	3.70	4.1	89.2	≥100
29	47.51	39.19	8.32	9.1	98.4	≥100
29	48.82	47.99	0.82	0.9	99.3	≥100
29	48.70	48.91	0.00	0.0	99.3	≥100

*: Calculated as the ratio between CO₂ produced (cumulative) and the ThCO₂ of sodium acetate: 85.5 mg CO₂/2 L

Table 8: CO₂ production and percentage biodegradation of the toxicity control

Day	HCL (0.05 N) titrated ml		Produced CO2(mL HCL)	Produced CO2(mg)	Cumulative CO2(mg)	Degradation* (%)
	Blank (mean)	Toxicity control
0	-	-	-	-	-	0
2	43.51	34.32	9.19	10.1	10.1	6
5	43.81	25.81	18.00	19.8	29.9	17
7	44.67	35.17	9.50	10.5	40.4	23
9	45.81	39.48	6.33	7.0	47.3	27
14	44.14	33.76	10.38	11.4	58.7	34
19	45.34	36.71	8.63	9.5	68.2	39
23	46.14	39.13	7.01	7.7	75.9	44
27	39.26	33.77	5.49	6.0	82.0	47
29	47.51	34.17	13.34	14.7	96.6	56
29	48.82	45.42	3.40	3.7	100.4	58
29	48.70	48.54	0.16	0.2	100.5	58

*: Calculated as the ratio between CO₂ produced (cumulative) and the sum of the ThCO₂ of the test substance and positive control: 172.7 mg CO₂/2 L; ThCO2 test substance: 87.2 mg CO₂/2 L; ThCO2 sodium acetate: 85.5 mg CO₂/2 L

Table 9: pH values of the different test media

	Just before the start of the test	On day 28
Blank control (A)	7.6	7.7
Blank control (B)	7.6	7.6
Positive control	7.6	8.1
TESAC (A)	7.6	7.6
TESAC (B)	7.5	7.5
Toxicity control	7.6	8.0

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance is readily not biodegradable since a 9% degradation (mean) was observed after 28 days (10-day window not passed). Based on the toxicity control performed within the study the test substance is not considered to be toxic to microbacteria.

Executive summary:

The aim of the study was to evaluate the test substance for its ready biodegradability (CO2 evolution test) in an aerobic aqueous medium with microbial activity introduced by inoculation of the supernatant of activated sludge. The procedure followed the OECD guideline No. 301 B. The positive control substance was degraded at least 60% (84%) within 14 days. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (34 mg CO2 per 2 L of medium). Within the test period the test item was degraded up to a maximum of 12%. Thus, the criterion for ready biodegradability (at least 60% biodegradation was not reached within ten days of biodegradation exceeding 10%) was not met. The difference of duplicate values for %-degradation of the test item was always less 20. In the toxicity control more than 25% degradation occurred within 14 days (34%). Therefore, the test substance was assumed to be not inhibitory on microbial activity. Since all criteria for acceptability of the test were met, this study is considered to be valid. In conclusion, the test item was not readily biodegradable and showed no inhibitory effects on the microbial activity under the performed test conditions.

Description of key information

not readily biodegradable (9% after 28 days; 10-d window not passed)

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater