2,4-Thiophenedicarbonitrile, 5-amino-3-methyl-

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:

10 November 2009 to 16 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

EC Number:

610-868-8

Cas Number:

52603-48-2

Molecular formula:

C7N3H5S

IUPAC Name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 16 November 2009 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: NDA
- Method of cultivation: NDA
- Storage conditions: NDA
- Storage length: NDA
- Preparation of inoculum for exposure: The activated sewage sludge sample 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. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C 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 deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C 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 4.0 g/l prior to use.
- Pretreatment: NDA
- Concentration of sludge: NDA
- Initial cell/biomass concentration: NDA
- Water filtered: yes - reverse osmosis purified and deionised
- Type and size of filter used, if any: Elga Optima 15+ or Elga Purelab Option R-15 BP

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

19.4 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

10 mg/L

Based on:

other: carbon content of test material

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: As recommended in the OECD guideline
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 litre (final volume) of reverse osmosis purified and deionised water, was added the following volumes of solutions a-d
Solution a 10 ml
Solution b 1 ml
Solution c 1 ml
Solution d 1 ml

- Additional substrate: Not used
- Solubilising agent (type and concentration if used): Not used
- Test temperature: approximately 21 °C
- pH: 7.6 - 7.7
- pH adjusted: no
- CEC (meq/100 g): NDA
- Aeration of dilution water: continuous; CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute
- Suspended solids concentration: final concentration of 30 mg suspended solids (ss)/l
- Continuous darkness: yes
- Other: stirred continuously by magnetic stirrer

TEST SYSTEM
- Culturing apparatus: 5 litre glass test vessels (containing 3 litres of solution)
- Number of culture flasks/concentration: 2, in inoculated culture medium, concentration = 19.4 mg/l substance (equivalent to 10 mg/l carbon)
- Method used to create aerobic conditions: CO2 free air was generated by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb(R)) granules.
- Method used to create anaerobic conditions: N/A
- Measuring equipment:
CO2 analysis
Samples (2 ml) were taken from the control, standard and test material first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, standard and test material and on Day 0 for the toxicity control. All samples were analysed 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 analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-Vcsh TOC analyser. Samples (300 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 to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
Samples (20 ml) were removed from all culture vessels on Day 0 and from the control, standard and test material culture vessels on Day 28 and filtered through Gelman 0.45 pm AcroCap filters (approximately 5 ml discarded) prior to DOC analysis.

The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 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 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 standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

- Details of trap for CO2 and volatile organics if used: 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
- Sampling frequency:
Samples for CO2 analysis (2 ml) were taken from the control, standard and test material first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, standard and test material and on Day 0 for the toxicity control.
Samples for DOC analysis (20 ml) were removed from all culture vessels on Day 0 and from the control, standard and test material culture vessels on Day 28.
- Sampling method: NDA
- Sterility check if applicable: NDA
- Sample storage before analysis: All samples were analysed for CO2 immediately.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (in duplicate)
- Abiotic sterile control: No
- Toxicity control: Yes

The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test material did not adsorb to filter matrices or to 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 the loss of any test material.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 38.38mg/l and therefore satisfied the validation criterion given in the OECD test guideline.
The IC content of the test material suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD test guideline.
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 guideline.
The toxicity control attained 42% degradation after 14 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.
Sodium benzoate attained 78% degradation after 14 days and 79% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

11

Sampling time:

28 d

Details on results:

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 material.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels with the exception of standard material R2. 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 material attained 11% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The toxicity control attained 42% degradation after 14 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 78% degradation after 14 days and 79% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC) gave percentage degradation values of -8% and -11 % respectively for the test material Replicates R1 and R2. Negative values were considered to be due to sampling/analytical variation.

Sodium benzoate attained 100% degradation for both Replicates R1 and R2 calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring.

Observations made throughout the test period showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved material visible.

BOD5 / COD results

Results with reference substance:

See table below

Any other information on results incl. tables

Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
2	46	2	21
6	74	0	38
8	70	0	37
10	69	0	39
14	78	5	42
21	78	13	‑
28	71	9	‑
29*	79	11	‑

- = No degradation result obtained due to toxicity control being terminated after 14 days.

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see above

Interpretation of results:

other: not readily biodegradable

Conclusions:

The test material attained 11% 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:

In an assessment of ready biodegradability study, CO2 evolution test (0656/0418), the test material was determined to not be readily biodegradable according to OECD Guideline 301B given that the degradation rates calculated from CO2 evolution values were less than 60 %.