Poly(oxy-1,2-ethanediyl), α-[2-(dide- cylmethylammonio)ethyl]- .omega.- hydroxy-, propanoate (salt) (Bardap 26)

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:

2003-12-15 to 2004-02-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with CO2 Evolution (Modified Sturm) test, Procedure C4-C 'CO2 Evolution test' and the Annex to Commission Directive 92/69/EEC, OECD Procedure 301B and the US Environmental Protection Agency (EPA), Office of Prevention, Pesticides and Toxic Substance (OPPTS) Method 835.3110 (m) (adopted January 1998).

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No information provided.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A sample of activated sludge was collected from Oakley sewage treatment works, which treats predominantly domestic wastewaters. Aliquots (25 mL) of a homognised sample were filtered through dried (approximately 105°C) and pre-weighed Whatman GFC filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solids concentration of 30 mg/L.

Duration of test (contact time):

>= 29 d

Initial conc.:

0.2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Remarks:

Mean cumulative 14CO2 production

Details on study design:

On the day before test initiation, ultrapure water (2.4 L) that had been aerated for thirty minutes then allowed to stand at room temperature for atleast 20 hours was added to each of eight five litre clear glass culture bottles that had been pre-treated with dimethyldichlorosilane (DMDCS). The bottles were covered with black polythene and volumes of each of the stock solutions required to prepare 3 litres of mineral salts medium were added. The bottles were inoculated then aerated overnight at a test temperature (22+/-2°C) with oil free and CO2-free compressed air that had been passed through cylinders containing fused calcium chloride and Carbosorb AS.

The supplied ethanol solution containing test substance, along with the cold standard solution of test substance, was analysed before test initiation, to determine the radiochemical content and purity.

On day 0 (at test initiation), an appropriate volume of test substance was added directly to the test cultures and to volumetric flasks containing ethanol which were to be used to assess the level of radioactivity applied. Ethanol was added at the same volume to the controls to be used for radiochemical analysis for consistency. The cultures were connected to a series of three Dreschel bottles, which contained 0.05 N sodium hydroxide solution.

The reference substance sodium benzoate was added from an aqueous stock solution (nominally 1.72 g/l, equivalent to 1000 mgC/l) to a culture bottle that contained inoculated mineral salts medium alone. This sample was used to assess biodegradative activity of the inoculum. The cultures were connected to a series of three Dreschel bottles, which contained 0.025 N barium hydroxide solution.

The pH of the cultures was measured at test initiation (day 0) and adjusted to 7.4 +/- 0.2 using 5N HCl. The contents were continually aerated with carbon dioxide free air and stirred.

The Total Viable Count (TVC) of a sample of inoculated medium was determined using the spreadplate method on Day 0. Aseptic technique was used to establish a suitable dilution series in sterile 1/4 strength Ringers solution and then aliquots (0.1 mL) were spread onto the surface of Yeast Extract Agar plates. The plates were incubated at 22+/-2°C for three days and the number of colony forming units that appeared on the plates containing between 30 and 300 colonies were used to calculate the TVC of the sample.

The Dreschel bottles nearest to the cultures were removed and radioactivity or carbon dioxide measurements at intervals for the duration of the test. Adjacent Dreschel bottles in the series were moved towards the culture bottle on each sampling occasion, and a new Dreschel bottle containing sodium hydroxide or barium hydroxide as appropriate, connected to the end of the series on each occasion. Duplicate samples from each sodium hydroxide trap were analysed by LSC to determine the radioactivity content.

The concentration of CO2 in the barium hydroxide traps was determined by titration with 0.05N hydrochloric acid, using phenolphthalein as indicator.

On day 28 of the test, titrations were undertaken and the pH of each culture determined. Concentrated hydrochloric acid (1 ml) was then added to each vessel to drive off dissolved inorganic carbon. The contents of the test vessels were aerated overnight and the final titrations carried out on Day 29.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

No preliminary study conducted.

Test performance:

Cumulative levels of CO2 production in the blank-controls after 29 days (73.7 and 78.1 mgCO2) were within the acceptable range for this assay system. This results confirm that the inoculum was viable and that the test was valid.

Parameter:

% degradation (CO2 evolution)

Remarks:

Mean cumulative 14CO2 production

Value:

1.6

Sampling time:

1 d

Parameter:

% degradation (CO2 evolution)

Remarks:

Mean cumulative 14CO2 production

Value:

10.8

Sampling time:

4 d

Parameter:

% degradation (CO2 evolution)

Remarks:

Mean cumulative 14CO2 production

Value:

19.9

Sampling time:

8 d

Parameter:

% degradation (CO2 evolution)

Remarks:

Mean cumulative 14CO2 production

Value:

25.9

Sampling time:

14 d

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Mean cumulative 14CO2 production

Value:

34

Sampling time:

29 d

Details on results:

The total viable count of a control culture on Day 0 of the test (1.3 x 10e8 cells/litre) was considered to be typical of the concentration of inoculum used in this type of test.

Cumulative CO2 production in the controls (73.7 and 78.1 mgCO2) was within the acceptable range for this assay system (recommended maximum for a three litre culture = 120 mgCO2). The degradation of sodium benzoate was rapid and had achieved 65% of its TCO2 after 6 days and 84% after 29 days.

Mean cumulative 14CO2 production was equivalent to 10% of the applied level of radioactivity after 4 days. The rate slowed by Day 8 (ca. 20% of AR) and had achieved 34% of the applied level of radioactivity by the end of the test on 29 days.

The pH of each test and control mixture was 7.6 at the start of the test and ranged between 7.5 and 7.6 at the end.

The air flow during the test ranged from 45 to 80 ml/minute.

The temperature of a three litre volume of water held under test conditions ranged from 21.8°C to 23.2°C during the test period.

Results with reference substance:

The degradation of sodium benzoate was rapid and had achieved 65% of its TCO2 after 6 days and 84% after 29 days in the positive control.

The pH of each test and control mixture was 7.6 at the start of the test and ranged between 7.5 and 7.6 at the end.

The rate of air flow during the test ranged from 45 to 80 mL/min.

The temperature of a three-litre volume of water held under test conditions ranged from 21.8°C to 23.2°C during test period.

Cumulative levels of CO2 production in the blank-controls after 29 days (73.7 and 78.1 mgCO2) were within the acceptable range for this assay system. This results confirm that the inoculum was viable and that the test was valid.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The cumulative levels of 14CO2 production by mixtures containing 14C-radiolabelled N,N-didecyl-N-methylpoly (oxyethyl) ammonium propionate were equivalent to ca 10% of the applied level of radioactivity after 4 days and 34% by the end of the test on day 29.

Substances are considered to be readily biodegradable under this test conditions if 14CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. 14C-radiolabelled N,N-didecyl–N-methyl-poly(oxyethyl)ammonium Propionate cannot, therefore, be considered to be readily degradable under these test conditions.

The results obtained for the degradation of sodium benzoate (65% of its TCO2) after 6 days and 84% after 29 days) and for cumulative CO2 production by the control mixtures (73.7 and 78.1% mgCO2) fulfil the validity criteria for this test.

Executive summary:

The ready biodegradability of N,N-didecyl–N-methyl-poly(oxyethyl)ammonium Propionate was determined in a Ready biodegradability study conducted in accordance with OECD Guideline 301B, Directive 92/69/EEC Procedure C.4-C and U.S.EPA OPPTS 853.3110. Mineral salts inoculated with activated sludge were treated with 14C-N,N-didecyl-N-methyl-poly(oxyethyl)ammonium Propionate and incubated for 29 days, with mean cumulative ¹⁴CO₂ production measured. The positive control consisted of mineral salts medium inoculated with activated sludge and dosed with sodium benzoate (10 mg C/L) while the negative control consisted of mineral salts medium dosed with ethanol and mineral salts medium inoculated with activated sludge.

The mean cumulative ¹⁴CO₂ production was equivalent to 10% after 4 days and progressed rapidly until Day 8. The rate of biodegradation then slowed. 34% biodegradation had occurred by Day 29. In the controls, the percentage biodegradation was 65% after 6 days, 84% after 29 days and the rate of ¹⁴CO₂ production was 73.7 and 78.1 mg CO₂ after 29 days.

Under the strict terms and conditions of the OECD Test Guidelines for ready biodegradability the test substance cannot be termed readily biodegradable.

Description of key information

The key study was carried out in accordance with OECD Guideline 301B. Mineral salts inoculated with activated sludge were treated with 14C-Didecylmethylpoly(oxyethyl)ammonium Propionate (0.2 mg/L

starting concentration) and incubated for 28 days. 34% biodegradation based on CO2 production was achieved at the end of the study and the test substance is therefore not readily biodegradable.
A supporting study was carried out, also in accordance with OECD Guideline 301B. "Ready Biodegradability; CO2 Evolution Test". Samples of activated sludge mixed with culture medium were dosed Didecylmethylpoly(oxyethyl)ammonium Propionate, sodium benzoate or a mixture of both.
The results from the key and the supporting study indicate that the substance, Didecylmethylpoly(oxyethyl)ammonium Propionate, is not readily biodegradable.
A further supporting (non-GLP) study of inherent biodegradability was performed according to the Zahn-Wellens test procedure. No control substance and no blank values were reported. It should be noted that the starting concentration of 303 C/L (corresponding to approx. 534 mg test substance/L) considerably exceeded the 3-hour EC50 for the inhibition of respiration of activated sludge organisms (11 mg test substance/L) and toxicity is likely to have influenced the outcome of the study. Elimination of Didecyldimethylpoly(oxyethyl)ammonium propionate was 53% after 3h, indicating a high degree of adsorption. Elimination reached 80% after 28 days, representing the combined effects of physico-chemical elimination and biodegradation (23% and 57%, respectively). Didecyldimethylpoly(oxyethyl)ammonium propionate removal between the untreated influent and the treated effluent due to the combined effects of abiotic and biological processes was expected to exceed 50% in industrial waste water treatment works.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Ready and Inherent Biodegradability:

Ready Biodegradability:

The key study was carried out in accordance with OECD Guideline 301B.

The ready biodegradability of ¹⁴C-N,N-didecyl–N-methyl-poly(oxyethyl)ammonium propionate, dosed at 0.2 mg/L, was determined in a ready biodegradability study conducted according to OECD Guideline 301B. The mean cumulative¹⁴CO₂ production was equivalent to 10% after 4 days and progressed rapidly until Day 8. The rate of biodegradation then slowed. 34% biodegradation had occurred by the end of the study, but was less than the 60% required to qualify for classification as readily biodegradable. Under the strict terms and conditions of the OECD Test Guidelines for ready biodegradability N,N-didecyl–N-methyl-poly(oxyethyl)ammonium propionate cannot be termed readily biodegradable.

A supporting study was carried out according to the same procedure (OECD Guideline 301B, CO2 Evolution Test), with non radio-labelled test substance dosed at a concentration of 5 mg C-equivalent/L. Didecyldimethylpoly(oxyethyl) ammonium propionate showed 0% degradation at the end of the incubation, confirming the indication of the key study, i.e. that the test substance is not readily biodegradable. There was no evidence of interference due microbial inhibition in the toxicity control included in the study design (the test substance caused no significant suppression of the degradation of the reference substance) and a subsequent (non-standard) toxicity check showed no evidence of microbicidal activity based on total viable counts of the inoculum after exposure to the same concentration of didecyldimethylpoly(oxyethyl) ammonium propionate under incubation conditions identical to those of the biodegradability assessment.

Inherent biodegradability:

In a further supporting study, the behaviour of Didecyldimethylpoly(oxyethyl)ammonium Propionate

was investigated according to OECD Guideline 302B (Zahn-Wellens Test). Activated sludge suspended in a standard mineral salts medium was dosed with Didecyldimethylpoly(oxyethyl)ammonium propionate at 303 mg C-equivalent/L. It should be noted that this concentration considerably exceeds the 3 -hour EC50 for the inhibition of respiration of activated sludge microorganisms and toxicity may have influenced the outcome of the study. Based on the measured DOC concentrations in samples taken after 3h and on days 1, 7, 10, 15, 20 and 28, the % elimination and % biodegradation was calculated after correction for the corresponding blank values.

53% elimination was observed after 3h, indicating a high degree of adsorption. Elimination of didecyldimethylpoly(oxyethyl)ammonium propionate reached 80% after 28 days, representing the combined effects of physico-chemical elimination and biodegradation processes (23% and 57%, respectively). Didecyldimethylpoly(oxyethyl)ammonium propionate removal between the untreated influent and the treated effluent due to the combined effects of abiotic and biological processes was expected to exceed 50% in industrial waste water treatment works.

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