N,N,4-trimethylpiperazine-1-ethylamine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-06 to 2017-07-26

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC NO. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW160141
- Expiration date of the lot/batch: 2018-12-30
- Purity test date: 2016-07-19 (certificate of analysis release date)
- Purity: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

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 26 June 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Storage conditions: The washed sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed twice by settlement and re-suspension 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.
- Pretreatment: no
- Concentration of sludge: The suspended solids concentration was equal to 2.2 g/L prior to use
- Water filtered: yes, purified water (Reverse osmosis purified and deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP)

Duration of test (contact time):

29 d

Initial conc.:

15.9 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

IC (inorganic carbon)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines.
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
(In order to avoid having to prepare solution (d) immediately before use, one drop of concentrated HCl per liter was added as a preservative).
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.
- Additional substrate: no
- Test temperature: controlled room at temperatures of between 22 and 25°C
- pH: 7.5-7.7, the pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.
- pH adjusted: yes, the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution
- Aeration of dilution water: 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.
- Suspended solids concentration: 30 mg ss/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 liter test culture vessels each containing 3 liters of solution
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: 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.
- Measuring equipment: The samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer.
- 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 water.

SAMPLING
- Sampling frequency: 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 sampled on Days 0 and 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 replicates
- Abiotic sterile control: yes, 2 replicates
- Toxicity control: yes, 1 replicate

STATISTICAL METHODS:
Statistical analysis of the Day 29 IC values for the inoculum control and test item vessels was carried out using a Student’s t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Preliminary Investigational Work
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. 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 32.98 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 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.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Not Readily Biodegradable

Details on results:

- Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.
- The toxicity control attained 30% biodegradation after 14 days and 41% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.
- The criterion for ready biodegradability (at least 60% biodegradation within 10 days of biodegradation exceeding 10%) was not met.

Results with reference substance:

Sodium benzoate attained 67% biodegradation after 14 days and 78% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1 Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	52	0	29
6	68	0	37
8	70	0	40
10	61	0	38
14	67	0	30
21	69	0	34
28	84	0	40
29*	78	0	41

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

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

A 28-d ready biodegradability test (OECD 301B, modified Sturm test) using activated sludge from a predominantly domestic waste water treatment plant indicated that the test item was not readily biodegradable under the conditions of the test (initial concentration 15.9 mg/L). The test substance showed 0% biodegradation. The test substance did not inhibit microbial activity at the concentration used in the test. The results of the test can be considered reliable without restriction.

Executive summary:

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 OCSPP 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 temperatures of between 22 and 25 °C for 28 days.

The biodegradation 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

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

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-11-28 to 2002-02-14

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

December 1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 180601
- Expiration date of the lot/batch: 2002-10-25
- Purity: 99.2% (GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: At least 96 h in water

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands.
- Storage conditions: The sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (30-90 minutes) and the liquid decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
- Pretreatment: no
- Concentration of sludge: The suspended solids concentration was equal to 4.9 g/L in the concentrated sludge.
- Water filtered: yes, 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.:

19 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines.
Solution a: KH2PO4: 8.50 g/L
K2HPO4: 21.75 g/L
Na2HPO4.12H2O: 67.20 g/L
NH4Cl: 0.50 g/L
dissolved in 1L Milli-Q water, pH = 7.4 ± 0.2
Solution b: CaCl2.2H2O: 36.40 g/L dissolved in 1L Milli-Q water
Solution c: MgSO4.7H2O: 22.50 g/L dissolved in 1 L Milli-Q water
Solution d: FeCl3.6H2O: 0.25 g/L dissolved in 1 L Milli-Q water
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.
- Additional substrate: no
- Test temperature: controlled room at temperatures of between 22 and 23°C
- pH: 7.6-8.2, the pH of the test preparations was determined on Day 0 and on Day 28 using Phenolphtalein as pH-indicator.
- pH adjusted: no
- Aeration of dilution water: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel.
- Suspended solids concentration: 4.9 g SS/L in the concentrated sludge.
- Continuous darkness: no data

TEST SYSTEM
- Culturing apparatus: 2-L all-glass brown coloured bottles
- Number of culture flasks/concentration: 2
* test substance and inoculum: 2 replicates
* inoculum blank: 2 replicates
* positive control: 1 replicate
* toxicity control: 1 replicate
- Method used to create aerobic conditions: A mixture of oxygen (~21%) and nitrogen (~79%) was led through a bottle, containing 0,5 - 1 L 0,0125 M Ba(OH)2 solution to trap CO2. The CO2-free air was sparged through the scrubbing solutions at a rate of ~1-2 bubbles per second ( ~30-100 mL/min).
- Measuring equipment: CO2 evolution was determined through titration of the remaining Ba(OH)2 with 0.05 M standardized HCl.
- Details of trap for CO2 and volatile organics if used: 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.

SAMPLING
- Sampling frequency: every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day
- Sampling method: the absorber bottle closest to the incubation system was sampled each time, the second and third bottle were moved one position closer to the system and a new bottle was added at the end
- On the 28th day, pH of test suspensions was measured and 1 mL of concentrated HCl was added to each bottle. Bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, two replicates with only inoculum
- Toxicity control: yes, one replicate with test item, reference substance, and inoculum
- Procedure control: yes, 1 replicate with reference item and inoculum

Reference substance:

acetic acid, sodium salt

Test performance:

- In the toxicity control more than 25% degradation occurred within 14 days (based on ThCO2). Therefore, the test substance was assumed to be not inhibitory on microbial activity.
- The positive control substance was degraded at least 60% within 9 days.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 5

Sampling time:

28 d

Remarks on result:

other: Mean of test bottle A and B

Details on results:

- The criterion for ready biodegradability (at least 60% biodegradation within 10 days of biodegradation exceeding 10%) was not met.

Results with reference substance:

- Sodium acetate attained 69% biodegradation after 14 days and 86% biodegradation after 29 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

A 28-d ready biodegradability test (OECD 301B, modified Sturm test) using unadapted activated sludge from a predominantly domestic waste water treatment plant indicated that the test item was not readily biodegradable under the conditions of the test (initial concentration 19 mg/L). The test substance showed 5% biodegradation. The test substance did not inhibit microbial activity at the concentration used in the test. The results of the test can be considered reliable without restriction.

Executive summary:

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 OCSPP 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 temperatures of between 22 and 25 °C for 28 days.

The biodegradation 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

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

Description of key information

Two studies (Desmares-Koopmans, 2002 and Best, 2017) were considered relevant to cover this endpoint and regarded as high quality studies (Klimisch score of 1).  The biodegradability of the test item was determined by Desmares-Koopmans (2002) according to OECD Guideline 301B and EU Method C.4 -C. using activated sludge from a predominantly  domestic waste water treatment plant. The study indicated that the test substance was not readily biodegradable under the conditions of the test (initial concentration 15.9 mg/L). The test substance showed 5% biodegradation.

In the second 28-d ready biodegradability test (OECD 301B, modified Sturm test) from Best (2017) the test item was also not readily biodegradable under the conditions of the test (initial concentration 19 mg/L). The test substance showed 0% biodegradation.

It can be concluded from both OECD 301B studies that the test substance is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed