Alcohols, C18-unsatd and ethoxylated C18-unsatd., phosphates (5 moles ethoxylation)

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:

From September 20, 2017 to November 02, 2017

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)

Deviations:

yes

Remarks:

The lab temperature slightly exceeded the 24°C upper tolerance limit on Day 28 of the study. However, the study author concluded that this slight deviation did not affect the test outcome.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

natural sediment: freshwater

Details on inoculum:

Surface water was collected from Malmesbury Pond, Sandymoor, Runcorn on 15th September 2017 and assigned a batch number of SE17002. It was filtered to remove coarse particles and aerated at 22 ± 2ºC prior to use. The surface water was then added to test media so as to achieve a final concentration of 100 mL per litre of test media. The test suspensions were then aerated until the study pre-conditioning period was ready to commence on 20th September 2017.

Duration of test (contact time):

ca. 42 d

Initial conc.:

ca. 14.8 mg/L

Based on:

test mat.

Initial conc.:

ca. 29.6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test medium
Test medium was prepared according to the method detailed in Cheshire Eco Solutions SOP III.19.

Test vessels
Conical flasks containing 3000 mL of test media.

Test and reference substances
Test material was added directly to the test vessels after being accurately weighed onto glass cover slips to give a final test concentrations of 14.8 mg/L and 29.6 mg/L. A control vessel was run in parallel, this contained inoculum but no test or reference material. A test vessel containing 102.9 mg (20 mg C/L) of the reference material, sodium benzoate (Batch No: 304526) in 3 litres of test media, was also included in the test. In addition a vessel containing 102.9 mg (20 mg C/L) of the reference material, sodium benzoate (Batch No: 304526) and test material at 29.6 mg/L in 3 litres of test media, was also included as an inhibition control.

Test conditions
3 L volumes of test media were stirred constantly in round, flat bottomed flasks in the dark. Carbon dioxide free air was passed through the test solutions at an estimated rate of 30 to 100 mL per minute. This was in turn passed through two dreschel bottles, per test vessel, containing barium hydroxide of known concentration and volume. The tests were carried out in the laboratory at 22 ± 2 deg C. Single vessels were prepared for each test material concentration.

Measurements
Measurements were taken after the appearance of a precipitate of barium carbonate, this was on Days 2, 4, 7, 11, 14, 19, 23, 28, 35, 42 and 43 (post acidification). The contents of each dreschel bottle were titrated against standardised hydrochloric acid to a pH 7 endpoint. Titres were taken for the barium hydroxide at the start and end of each time period.

Analysis of study data
Based on the titration values the quantities of carbon dioxide evolved over each incubation interval were determined based on the relationship of 1mL of titre being equivalent to 0.3 mg carbon. Since 1 mmol of CO2 is produced for every mmol of barium hydroxide reacted to barium chloride and 2 mmol of HCl are needed for the titration of the remaining barium hydroxide, and given that the molecular weight of CO2 is approximately 44 g, the weight of CO2 produced (mg) is calculated by:

0.1 x (Start titre [mL] - HCl titrated [mL]) x 44 / 2 = 2.2 x (Start titre [mL] - HCl titrated [mL]

Thus, the factor to convert volume of 0.1M HCl titrated to mg CO2 is 2.2. The percentage degradation of the test substance was determined from the ratio of total carbon evolved to carbon present, based on the carbon content of test substance of 61.3% w/w. This analysis was conducted by Scymaris Ltd, an outside contract laboratory, who are members of the UK GLP compliance monitoring programme.

The percentage biodegradation is calculated from:
% degradation = mg CO2 produced / (mg TOC added in test x 3.67)

where, 3.67 is the conversion factor (44/12) for carbon to carbon dioxide.

Reference substance:

benzoic acid, sodium salt

Remarks:

20 mg C/L

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

14.8 mg/L test substance

Value:

ca. 44.8

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

29.6 mg/L test substance

Value:

ca. 9.1

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

14.8

Value:

ca. 84

Sampling time:

42 d

Remarks on result:

other: inherently biodegradable

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

29.6 mg/L

Value:

ca. 12.7

Sampling time:

42 d

Details on results:

A figure of 60% degradation within 28 d is usually taken as being indicative of a good potential for degradation. According to this guideline, under these test conditions in the OECD 301B Procedure, test substance showed low potential for degradation at the test concentration of 29.6 mg/L and limited potential at 14.8 mg/L after 28 d in this study. The study was extended up to 42 d after which time test substance showed good potential for degradation at concentration 14.8 mg/L and low potential at 29.6 mg/L concentration. The final 42-d cumulative % degradation values were determined to be 84% at 14.8 mg/L and 12.7% at 29.6 mg/L.

Results

Table 1: Mean titration values (mL) for barium hydroxide samples neutralised with 0.1 M hydrochloric acid in study CES170601

Day of titration	Control	Sodium benzoate (34.3 mg/L)	Test substance (14.8 mg/L)	Test substance (29.6 mg/L)	Inhibition control*
2	11.7	35.1	13.1	16.8	33.4
4	7.8	19.7	9.1	6.6	21.3
7	6.5	20.4	12.6	5.7	26.7
11	17.7	24.3	17.4	15.5	24.9
14	11.9	18.9	13.5	15.3	20.4
19	7.4	9.6	9.5	9.2	14.4
23	4.3	5.1	7.6	7.2	15
28	12.4	13	19.1	12.3	8.6
35	7.5	9.8	17.7	9.8	14.5
42	4.8	3.2	12.6	7.4	8.5
43	5.4	5.1	7	4.1	8

*Inhibition control contained sodium benzoate at 34.3 mg/L and test substance at 29.6 mg/L in 3 L of test media

The Table 1 values are the difference between the titres at the start and end of each time period.

Table 2: Evolved carbon dioxide values (mg) due to test or reference material in study CES170601

Period	Control	Sodium benzoate (34.3 mg/L)	Test substance (14.8 mg/L)	Test substance (29.6 mg/L)	Inhibition control*
2	25.7	77.7	28.8	37	73.5
4	17.2	43.3	20	14.5	46.9
7	14.2	44.9	27.7	12.5	58.7
11	38.8	53.5	38.3	34.1	54.8
14	26.1	41.6	29.7	33.7	44.9
19	16.3	21.1	20.9	20.2	31.7
23	9.5	11.2	16.7	15.8	33
28	27.2	28.6	42	27.1	18.9
35	16.4	21.6	38.9	21.6	22.2
42	10.6	7	27.7	16.3	18.7
43	11.9	11.2	15.4	9	17.6

*Inhibition control contained sodium benzoate at 34.3 mg/L and test substance at 29.6 mg/L in 3 L of test media

Table 3: Cumulative biodegradation

Cumulative degradation (%)
Day of titration	Sodium benzoate (34.3 mg/L)	Test substance (14.8 mg/L)	Test substance (29.6 mg/L)	Inhibition control*
2	23.4	2.8	5.1	21.7
4	35.3	5.4	3.9	35.2
7	49.2	17.7	3.1	55.4
11	55.8	17.2	1	62.6
14	62.9	20.5	4.4	71.2
19	65.1	24.7	6.2	78.2
23	65.9	31.3	9.1	88.9
28	66.5	44.8	9.1	85.1
35	68.9	65.2	11.5	87.8
42	67.3	80.8	14	91.5
43	67	84	12.7	94.1

*Inhibition control contained sodium benzoate at 34.3 mg/L and test substance at 29.6 mg/L in 3 L of test media

Comments

A degradation figure of 62.9% after 14 d was obtained for the reference material. This demonstrates that the inoculum was biologically active. A degradation figure of 71.2% after 14 d was obtained for the inhibition control. This demonstrates that the inoculum was biologically active in the presence of test substance at the higher test concentration of 29.6 mg/L. CO2 production the Control vessels was 71.2 mg/L of test media in this study. This was within the acceptable limits defined in the method.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the study conditions, the test substance was determined to be not readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd AE5 PSE', according to OECD Guideline 301B, using modified sturm test, in compliance with GLP. The test substance was assessed for the rate and extent of biodegradation when exposed to freshwater microorganisms over a period of 42 d. Test substance was added directly to the test vessels after being accurately weighed onto glass cover slips to give a final test concentrations of 14.8 mg/L and 29.6 mg/L. A control vessel was run in parallel, this contained inoculum but no test or reference material. A test vessel containing 20 mg C/L of the reference material, sodium benzoate in 3 L of test media, was also included in the test. In addition a vessel containing 20 mg C/L of the reference material, sodium benzoate and test substance at 29.6 mg/L in 3 L lof test media, was also included as an inhibition control. 3 L volumes of test media were stirred constantly in round, flat bottomed flasks in the dark. Carbon dioxide free air was passed through the test solutions at an estimated rate of 30 to 100 mL per minute. This was in turn passed through two dreschel bottles, per test vessel, containing barium hydroxide of known concentration and volume. The tests were carried out in the laboratory at 22 ± 2ºC. Single vessels were prepared for each test substance concentration. Measurements were taken after the appearance of a precipitate of barium carbonate, this was on Days 2, 4, 7, 11, 14, 19, 23, 28, 35, 42 and 43 (post-acidification). The contents of each dreschel bottle were titrated against standardised hydrochloric acid to a pH 7 endpoint. After 28 d, the test substance showed low potential for degradation at 29.6 mg/L and a limited potential at 14.8 mg/L. This test was extended up to 42 d, after which time test substance showed good potential for degradation at the test concentrations of 14.8 mg/L and 29.6 mg/L. The final 42-d cumulative % degradation values were determined to be 84% at 14.8 mg/L and 12.7% at 29.6 mg/L. A degradation figure of 62.9% after 14 d was obtained for the reference material. This demonstrates that the inoculum was biologically active. A degradation figure of 71.2% after 14 d was obtained for the inhibition control. This demonstrates that the inoculum was biologically active in the presence of test substance at the higher test concentration of 29.6 mg/L. CO2 in the Control vessels was 71.2 mg/L of test media in this study. This was within the acceptable limits defined in the method. Therefore, the study had met all the validity criteria. Under the study conditions, the test substance was determined to be not readily biodegradable (Cheshire, 2018). However, given the >20% degradation of the test substance at lower test concentration within 28 d, it may be regarded as showing evidence of inherent primary biodegradability (OECD, 2005).

Description of key information

Based on the study results, the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE' can be considered to be inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

A study was conducted to determine the ready biodegradability of the test substance, ‘mono- and di- C18-unsatd PSE and C18-unsatd AE5 PSE’, according to OECD Guideline 301B, using modified sturm test, in compliance with GLP. The test substance was assessed for the rate and extent of biodegradation when exposed to freshwater microorganisms over a period of 42 d. Test substance was added directly to the test vessels after being accurately weighed onto glass cover slips to give final test concentrations of 14.8 mg/L and 29.6 mg/L. A control vessel was run in parallel, this contained inoculum but no test or reference material. A test vessel containing 20 mg C/L of the reference material, sodium benzoate in 3 L of test media, was also included in the test. In addition a vessel containing 20 mg C/L of the reference material, sodium benzoate and test substance at 29.6 mg/L in 3 L lof test media, was also included as an inhibition control. 3 L volumes of test media were stirred constantly in round, flat bottomed flasks in the dark. Carbon dioxide free air was passed through the test solutions at an estimated rate of 30 to 100 mL per minute. This was in turn passed through two dreschel bottles, per test vessel, containing barium hydroxide of known concentration and volume. The tests were carried out in the laboratory at 22 ± 2ºC. Single vessels were prepared for each test substance concentration. Measurements were taken after the appearance of a precipitate of barium carbonate, this was on Days 2, 4, 7, 11, 14, 19, 23, 28, 35, 42 and 43 (post-acidification). The contents of each dreschel bottle were titrated against standardised hydrochloric acid to a pH 7 endpoint. After 28 d, the test substance showed low potential for degradation at 29.6 mg/L and a limited potential at 14.8 mg/L. This test was extended up to 42 d, after which time test substance showed good potential for degradation at the test concentrations of 14.8 mg/L and 29.6 mg/L. The final 42-d cumulative % degradation values were determined to be 84% at 14.8 mg/L and 12.7% at 29.6 mg/L. A degradation figure of 62.9% after 14 d was obtained for the reference material. This demonstrates that the inoculum was biologically active. A degradation figure of 71.2% after 14 d was obtained for the inhibition control. This demonstrates that the inoculum was biologically active in the presence of test substance at the higher test concentration of 29.6 mg/L. CO2 in the Control vessels was 71.2 mg/L of test media in this study. This was within the acceptable limits defined in the method. Therefore, the study had met all the validity criteria. Under the study conditions, the test substance was determined to be not readily biodegradable (Cheshire, 2018). Given the low solubility of the substance (based on a CMC of 44 mg/L), the observed lower degradation rate at the higher concentration could be attributed to the formation of micelles, leading to lesser bioavailability of the test substance to the microbes. In addition, the lower biodegradation potential could be attributed to the use of only surface water as inoculum, instead of an inoculum from a waste water treatment plant, which is known to have much more "degradation power" than just surface water.

In light of these above facts, together with the >20% degradation of the test substance at lower test concentration within 28 d, the test substance may be regarded as showing evidence of inherent primary biodegradability (OECD, 2005).

[Type of water: freshwater]