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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study performed on propanamine salt (ammonium salt more likely to be biodegradable)
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
the test substance was added in portions (20% on Day 0, 30% on Day 1 and 50% on Day 2)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge was obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachusetts and the New Bedford Wastewater Treatment Plant, New Bedford, Massachusetts. These treatment facilicties receive primarily domestic waste.
Approximately 4 liters of acitvated sludge was collected on 9 February 2005 from each plant, and transported to Springborn Smithers. Upon arriveal at Springborn Smithers, the sludge was passed through a 2-mm sieve, combined, and centrifuged at 1500 rpm fopr 10 minutes. The supernatant was discarded, the sludge was washed with tap water and the contents were centrifuged at least once again and the supernatant was discarded. The moisture content of the activated sludge was determined, using an automated moisture analyzer (Sartorius MA-30), to be 94.6% and the percent solids determined to be 5.36%. A 15 mg solids/mL inoculum solution was prepared (55.97 g wet sludge with 200 mL mineral medium) and aerated until used.
Both test substance flasks, the blank flasks, the procedural control flask, and the toxicity control flasks all received 6.0 mL of the inoculum to produce an activated sludge concentration of 30 mg/L.
In addition, a 100-g aliquot of fresh soil was collected on 10 Febraury 2005 in a wooded area near Springborn Smithers Laboratories. Upon arrival at Springborn Smithers, the soil was suspended in 1 L of tap water. The suspesion was filetered through glass wool and refrigerated until use. A 3 mL aliquot of the soil filtrate and 3 mL of Weweantic River water was added to each vessel containing 3 L of mineral medium.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
other: TOC
Initial conc.:
16.7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium(according to guideline)
- Additional substrate: test substance (at 10 mg/L organic carbon), reference substance (at 10 mg/L organic carbon) and a mixture of both (at 20 mg/L organic carbon (10 mg/L of both substances)). Test substance concentration of 10 mg/L organic carbon is equivalent to 16.7 mg/L Biosoft N 411,based on purity of 91.9% and percentage C 65.8% based on the molecular formula of the test substance.
- Solubilising agent (type and concentration if used): not used
- Test temperature: 21.8-22.0°C
- pH: Start: 7.52; Day 28: 7.35 - 7.50
- pH adjusted: no.
- Suspended solids concentration: to each reactor activated sludge was added corresponding to 30 mg suspended solids/L. Additionally 1 ml/L soil filtrate and 1 ml/L Weweantic River water were added with unknown solids content
- Continuous darkness: yes
- Other: the test vessels were sealed and CO2-free air bubbled through the solution and stirred continuously by magnetic stirrers

TEST SYSTEM
- Culturing apparatus: 4-Liter test culture vessels each containing 3 litres of solution. The test vessels were sealed and CO2-free air bubbled through the solution and stirred continuously by magnetic stirrers. The CO2 produced by degradation was collected in two CO2 effluent gas traps, the first containing 200 ml of 0.2 N Potassium hydroxide (KOH) and the second trap containing 100 ml of KOH. The CO2 absorbing solutions were prepared using purified water with a DOC content <0.5 ppm.
- Number of culture flasks/concentration: 2 with test substance, 2 with only inoculum (blank),1 with reference substance and 1 as toxicity control with combined test substance and reference substance
- Method used to create aerobic conditions: The test system was aerated with CO2-free air
- Measuring equipment: On test days 2, 4, 7, 10, 14, 19 and 24, a 3 mL sample was removed from the first KOH carbon dioxide trap on each test system and analyzed for CO2 evolution. On test day 28, samples for analysis of CO2 evolution were also removed from the second trap. The amount of evolved CO2 in each trap was determined using a ThermoGlas Model 1200 Carbon Analyzer. On day 28, 1 mL of concentrated HCl was added to the reactor flasks to terminate biological activity. Aeration was continued overnight to drive off any inorganic carbon from the test vessels.
- Details of trap for CO2 and volatile organics if used: see above
- Test procedure: Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2994 mL of mineral medium and 6 mL of activated sludge inoculum, 3 mL of soil filtrate and 3 ml of Weweantic River water and aerated with CO2-free air overnight. Over the first three test days, the test substance and toxicity control replicate vessels were dosed incrementally with a total of 30 mL (i.e., 6.0, 9.0 and 15 mL on days 0, 1 and 2, respectively) of the 1.0 mg C/L Biosoft N 411 stock solution.The total fortification was 10 mg C/L in the test substance vessels. At test initiation, the toxicity control vessel also received 3.0 mL of the 10.0 mg C/L sodium benzoate stock solution, The total fortification was 20 mg C/L (10 mg C/L test substance and 10 mg C/L reference substance) in the toxicity control vessel. The sodium benzoate procedural control was fortified with 3.0 mL of the sodium benzoate stock solution for a final concentration of 10 mg C/L.
DOC samples were not taken at test initiation because of the incremental dosing procedure followed during the first three days.
SAMPLING
- Sampling frequency: day 2, 4, 7, 10, 14, 19, 24 and 28
- Sampling method: Samples (3 mL) were taken from the first CO2 absorber vessels. The second absorber vessels were all sampled on Day 28. Samples were analyzed for CO2 immediately. On day 28, 1 mL concentrated hydrogen chloride acid (HCl) was added to each vessel to terminate biological activity. The vessels were resealed, aerated overnight to drive any residual inorganic carbon form the test vessels.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (in duplicate)
- Toxicity control: reference substance and test substance combined
- Other: reference substance used was sodiumbenzoate

CALCULATIONS:
-The amount of carbon dioxide evolved from each test system was adjusted by subtracting the CO2 production value from the blank control.
The percentage degradation for each test system is calculated using the following equation and is expressed as cumulative percent biodegradation (or percent of theoretical CO2 production):
% degradation = ((mg CO2 produced)/ (mg TOC added as test chemical x 3.67)) x 100
where: The molecular weight conversion factor for carbon to carbon dioxide is 3.67
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
87.35
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
81.21
Sampling time:
10 d
Results with reference substance:
Sodium benzoate attained 77.2% degradation after 10 days and 84.2% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

The toxicity control attained 80.9% degradation after 10 days and 86.3% degradation after 28 days thereby confirming that the test item was not toxic to the micro-organisms used in the test.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1) , was at 87.35% degraded in 28 days. The substance is considered readily biodegradable.
Executive summary:

The biodegradability of benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1) (LASIPA)was studied in a OECD 301 B (CO2 -evolution) test. The test material was Biosoft N 411. Tests were performed with 10 mg organic carbon/L, equivalent to 16.7 mg/L Biosoft N 411, added in 3 doses over the first days (20% ond Day 0, 30% on Day 1 and 50% on Day 2),to flasks containing activated sludge, supplemented with soil filtrate and river water. The CO2 production was then measured as inorganic carbon captured in gas wash flasks filled with 0.025 M KOH over the next 28 days.

Sodium benzoate was used as a reference substance and as toxic control both the test substance and the reference substance were combined. Sodium benzoate attained 77.2% degradation after 10 days and 84.2% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The toxicity control attained 80.9% degradation after 10 days and 86.3% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. The 10-day window validation criterion does not apply for UVCB substances. The test substance, LASIPA, is readily biodegradable.

Description of key information

The biodegradability of Benzenesulfonic acid, C10-13-alkyl derivs., compds. with 2-propanamine (1:1) was studied in a OECD 301 B (CO2 -evolution) test.

The test substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The biodegradability of Benzenesulfonic acid, C10-13-alkyl derivs., compds. with 2-propanamine (1:1) was studied in a OECD 301 B (CO2 -evolution) test. The test substance is readily biodegradable.