Glycerides, tall-oil mono-

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:

26. Feb. 2017 to 28. Mar. 2018

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:

OECD Guidelines for the Testing of Chemicals, Part 301 B, adopted 17. Jul. 1992 “CO2-Evolution-Test (Modified STURM Test)“

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation (EC) No. 440/2008, Method C.4-C, adopted 30. May 2008 “CO2-Evolution-Test”

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Specification
Activated sludge from a biologic sewage treatment plant was used as inoculum. The chosen plant treats mostly domestic sewage.

Source and Pre-Treatment of inoculum
Source
The sludge was taken from the activation basin of the sewage treatment plant in D-67480 Edenkoben.
Date of collection: 23. Feb. 2018, batch no: 20180223.

Pre-Treatment
The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was then aerated until use. The dry matter was determined to contain 3980 mg of suspended solids/L.

Duration of test (contact time):

28 d

Initial conc.:

> 27.7 - < 29.8 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparations
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its DOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.

Experimental Parameters
Flask volume: 1500 mL
Apparatus blanks: 2, containing mineral medium only
Blank Controls: 2, containing mineral medium and inoculum
Positive control flasks: 2, containing positive control, mineral medium and inoculum
Test flasks: 2, containing test item, mineral medium and inoculum
Abiotic control: 1, containing test item, mineral medium and HgCl2
Toxicity control: 1, containing test item, positive control, mineral medium and inoculum
Inoculum concentration: 25.0 mg/L
Temperature: 19.5 – 21.9 °C
Duration: 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L of the test item.

Apparatus
The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.
Magnetic stirrers were used to prevent deposition of inoculum.
The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.

Sampling
From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2 (on day 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.
On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2.
On day 29, samples from both scrubber flasks were taken.

Reference substance:

aniline

Test performance:

All validity criteria were met.
Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation was not detected. Both replicates of the test item showed good correspondence.
If degradation in the toxicity flask is below 25 % after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 68 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 27.7 mg/L”.
For pure substances ready biodegradability is defined in the guidelines as degradation surpassing 60 % within 10 days after reaching a level of 10 % (10 day-window).
Because the test item is a mixture, the 10 day-window has not to be taken into account. Therefore, the test item is considered as “readily biodegradable” within 28 days”.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

90

Sampling time:

28 d

Details on results:

The test item LUMULSE GMT-K is considered as “readily biodegradable“.
The degree of biodegradation reached 90 % after 28 days.
The 10-day-window began on day 2 (graphic evaluation), at its end, 70 % degradation were reached, surpassing the pass level of 60 % given in the OECD guideline.
Because the test item is a mixture, the 10 day-window has not to be taken into account. Therefore, regardless of the 10 day-window, the test item is considered as “readily biodegradable within 28 days”.
Abiotic degradation was not detected.

Results with reference substance:

Degradation of the positive control was 67 % after 11 days.

IC-Values

In the following tables, the IC values (given in mg/L) which were measured in the samples of the front scrubber flasks are stated.

IC values in mg/L of apparatus blanks, blank controls, front scrubber

Day	Apparatus blank 1	Apparatus blank 2	Blank Control 1	Blank Control 2
0	3.50	3.55	2.51	5.15
2	5.15	3.74	11.94	7.35
4	5.18	4.09	20.25	15.31
7	7.59	5.97	31.73	35.14
9	10.76	12.78	38.84	61.61
11	12.07	9.42	43.26	56.66
14	12.48	10.91	47.08	56.72
18	15.66	14.12	54.14	68.26
23	19.31	16.62	64.85	85.02
29	26.31	23.63	74.96	97.79

 

IC values in mg/L of positive control, test flasks, front scrubber

Day	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.65	2.89	3.02	2.84	2.57	2.95
2	12.91	21.55	30.59	35.14	6.12	26.47
4	18.96	91.53	109.89	120.60	7.60	153.80
7	141.56	198.45	211.16	197.34	9.83	354.80
9	232.76	229.82	257.57	227.42	19.64	298.24
11	258.64	259.40	296.73	256.91	12.14	444.05
14	275.92	278.57	314.19	268.39	13.18	483.80
18	321.19	319.50	362.45	318.06	16.19	571.44
23	381.82	348.82	394.10	353.72	19.56	*638.60
29	405.75	374.43	414.07	374.44	23.52	*675.62

*Because the calibration limit was exceeded the toxicity sample was diluted 1:2 in aqua demin.

 

In the following tables, the IC values which were measured in the samples of the backscrubber flasks are stated.

IC values in mg/L of blank controls, apparatus blanks, back scrubber

Day	Apparatus blank 1	Apparatus blank 2	Blank Control 1	Blank Control 2
0	2.62	2.74	2.39	3.27
29	4.30	4.26	3.84	5.09

 

IC values in mg/L of positive control, test flasks, back scrubber

Day	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.84	3.06	2.65	2.71	2.61	2.91
29	5.82	3.84	4.11	4.18	3.92	4.18

 

Net IC

For each flask, the net IC was calculated by subtracting the mean IC values of the apparatus blanks of the corresponding sampling date from the remaining IC values. Exception: Values of day 0 do not need to be corrected.

The value of day 0 of the apparatus blank was subtracted from the apparatus blanks of the corresponding sampling dates in advance.

The net IC values are presented in the following tables.

 

Net IC-values in mg/L of front scrubber flasks

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.5	5.2	2.7	2.9	3.0	2.9	2.6	3.0
2	11.0	6.4	12.0	20.6	29.7	34.2	5.2	25.6
4	19.1	14.2	17.9	90.4	108.8	119.5	6.5	152.7
7	28.5	31.9	138.3	195.2	207.9	194.1	6.6	351.5
9	30.6	53.4	224.5	221.6	249.3	219.2	11.4	390.0
11	36.0	49.4	251.4	252.2	288.5	249.7	4.9	436.8
14	38.9	48.6	267.8	270.4	306.0	260.2	5.0	475.6
18	42.8	56.9	309.8	308.1	351.1	306.7	4.8	560.1
23	50.4	70.6	367.4	334.4	379.7	339.3	58.1	624.2
29	53.5	76.3	384.3	353.0	392.6	353.0	2.1	654.2

 

Net IC-values in mg/L of back scrubber flasks

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.4	3.3	2.8	3.1	2.7	2.7	2.6	2.9
29	2.2	3.5	4.2	2.2	2.5	2.6	2.3	2.6

 

pH

in the following table, the pH at the end of the test (before addition of HCI) is given:

 

pH in Test flasks on day 28

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
28	7.4	7.4	7.7	7.7	7.5	7.5	7.0	7.6

 

Equations

Emitted Carbon in mg/L

Emitted carbon in mg/L test solution in the respective vessel at time t was calculated using the following equation:

(IC(t) – IC(0)) * VolNaOH(t)

emittC = -------------------------------------------------------

VolTestVessel

 

With

emittC            emitted carbon in mg/L test solution

IC(t)                net inorganic carbon in mg/L NaOH in the respective vessel at time t

IC(0)               net inorganic carbon in mg/L NaOH in the respective vessel at the start of the test

VolNaOH (t)   remaining volume NaOH in L in the scrubber at time t

                       (Volume at t = 0 (here: 0.1L) – Ʃ (all sample volumes up to time t))

VolTestVesseltest vessel volume in L (here: 1:5)

 

For day 29, the IC content of both scrubber flasks were taken into account.

Calculation of emitted carbon in necessary for the assessment of validity. The value obtained with this equation is multiplied with 3.667 (44/12) in order to obtain emitted CO₂.

 

Degradation in %

The percentage biodegradation in the test flasks was calculated from:

            emittedC (Test) in mg/L – Mean emittedC (Controls) in mg/L

% degradation = ----------------------------------------------------------------------------------------- *100%

added C in mg/L

 

Degradation in positive control and toxicity flasks was calculated analogously.

 

Abiotic degradation was calculated from:

                       mg/L emittedC (abiotic)

% degradation = ------------------------------------ *100%

added C in mg/L

 

Calculation Results

 

Emitted Carbon in mg/L

In the following table, calculated emitted carbon (from net IC and equation stated above) is presented.

 

Emitted carbon in mg/L

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
2	0.56	0.08	0.62	1.17	1.76	2.06	0.17	1.49
4	1.09	0.59	0.99	5.72	6.91	7.61	0.26	9.78
7	1.68	1.73	8.77	12.44	13.25	12.36	0.26	22.54
9	1.80	3.09	14.20	14.00	15.76	13.84	0.56	24.77
11	2.12	2.81	15.76	15.79	18.08	15.63	0.15	27.48
14	2.28	2.72	16.61	16.76	18.99	16.12	0.15	29.62
18	2.50	3.21	19.04	18.93	21.58	18.83	0.14	34.54
23	2.94	4.01	22.37	20.33	23.10	20.63	0.16	38.10
29	3.08	4.33	23.24	21.18	23.63	21.23	-0.05	39.49

 

Degradation Values

In the following table, the percentage biodegradation is presented:

Degradation values in %

Day	Positive Control 1	Positive Control 2	Positive Control Mean	Test 1	Test 2	Test Mean	Abiotic Control	Toxicity Control
2	1.5	4.2	2.9	6.8	8.7	7.7	0.9	2.9
4	0.8	24.4	12.6	28.6	33.7	31.1	1.3	22.5
7	35.3	53.6	44.5	54.3	53.0	53.6	1.3	52.4
9	58.7	57.7	58.2	62.7	56.7	59.7	2.8	56.2
11	66.4	66.6	66.5	73.5	65.4	69.4	0.7	62.9
14	70.5	71.3	70.9	77.6	67.7	72.6	0.8	68.2
18	80.9	80.3	80.6	88.1	79.4	83.8	0.7	79.7
23	94.4	84.2	89.3	92.3	85.3	88.8	0.8	87.1
29	97.6	87.3	92.4	93.7	87.1	90.4	-0.2	90.0

 

As the measured IC values in the abiotic control are very low, measurement uncertainties lead to negative degradation values while in fact no degradation has taken place.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item LUMULSE GMT-K is considered as “readily biodegradable“.
The degree of biodegradation reached 90 % after 28 days.
The 10-day-window began on day 2 (graphic evaluation), at its end, 70 % degradation were reached, surpassing the pass level of 60 % given in the OECD guideline.
Because the test item is a mixture, the 10 day-window has not to be taken into account.
Therefore, regardless of the 10 day-window, the test item is considered as “readily biodegradable within 28 days”.

Executive summary:

The test item LUMULSE GMT-K was tested using a concentration of nominally 20 mg organic carbon/L LUMULSE GMT-K in test medium following OECD 301B and EU-Method C.4-C.

 

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L).

The test was left running for 28 days.

All validity criteria were met. Degradation of the positive control was 67 % after 11 days.

 

The following data were determined for the test item LUMULSE GMT-K:

10-day-window:                                            day 2–12

degradation at the end of 10-day-window    70 %

degradation at the end of the test                 90 %

pass level following guideline:           60 % at the end of 10-day-window for pure substances

respective 60 % at the end of the test for mixtures

 

Therefore, when applying the 10-day-window, LUMULSE GMT-K is readily biodegradable following OECD 301B and EU C.4-C respectively.

 

Because the test item is a mixture the 10-day-window does not have to be taken into account.

As degradation surpassed 60% in the course of the test, LUMULSE GMT-K is considered as readily biodegradable.

Description of key information

LUMULSE GMT-K is considered as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The test item LUMULSE GMT-K was tested using a concentration of nominally 20 mg organic carbon/L LUMULSE GMT-K in test medium following OECD 301B and EU-Method C.4-C.

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L).

The test was left running for 28 days.

All validity criteria were met. Degradation of the positive control was 67 % after 11 days.

 

The following data were determined for the test item LUMULSE GMT-K:

10-day-window:                                            day 2–12

degradation at the end of 10-day-window    70 %

degradation at the end of the test                 90 %

pass level following guideline:           60 % at the end of 10-day-window for pure substances

respective 60 % at the end of the test for mixtures

 

Therefore, when applying the 10-day-window, LUMULSE GMT-K is readily biodegradable following OECD 301B and EU C.4-C respectively.

Because the test item is a mixture the 10-day-window does not have to be taken into account.

As degradation surpassed 60% in the course of the test, LUMULSE GMT-K is considered as readily biodegradable.

[Type of water: freshwater]