Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol]and propylidynetrimethanol and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with OECD guidelines and GLP standards.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Activated sludge was obtained from Totness Sewage Treatment works, Devon, the day before the start of exposure of test and reference substances. This works treats sewage of predominantly domestic origin.
The activated sludge was settled and the supernatent decanted. Sludge was washed by centrifugation for 5 minutes, the supernatent was decanted and made up to the original volume by addition of mineral media solution. The washing was repeated twice more and the sludge was aerated at room temperature until the it was used in the test.

Duration of test (contact time):

28 d

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

See methods below

Reference substance:

aniline

Test performance:

The biodegradation of aniline was greater than 70% after 14 days, confirming the viability of the activated sludge. The PMS probe was calibrated on day 0 and was found to be within range. Continuous monitoring of the room temperature was started on day 3 and showed that the temperature remained within the range of 22.5 ±2.5oC during the study. As such, the test should be considered to be valid.

Parameter:

% degradation (DOC removal)

Value:

92

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

89

Sampling time:

14 d

Details on results:

The degradation of the test substance was 89% after 14 days and 92% after 28 days. As biodegradtion of HB TMP was greater than 70% by day 14, the substance can be considered inherently biodegradable.

Results with reference substance:

The reference substance (aniline) was >70% degraded within 14 days, confirming the viability of the active sludge.

Table1: Test substance dissolved organic carbon results

Test time	Blank (mg/l)	Measured DOC (mg/l)	Blank corrected DOC (mg/l)	Biodegradation (%)
3h	20	247	227	-
3d	4.3	234	230	<1
7d	2.6	115	113	50
14d	5.7	32	26	89
21d	8.4	29	20	91
27d	9.0	27	18	92
28d	9.2	26	17	92

Table 2: Reference substance dissolved organic carbon results

Test time	Blank (mg/l)	Measured DOC (mg/l)	Blank corrected DOC (mg/l)	Biodegradation (%)
3h	20	288	268	-
3d	4.3	258	154	5.3
7d	2.6	14	11	96
14d	5.7	9.3	3.6	99
21d	8.4	12	3.3	99
27d	9.0	8.5	<1	100
28d	9.2	9.2	0.0	100

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The substance was 92% degraded after 28 days. As such, the substance can be considered to be inherently biodegradable.

Executive summary:

A 28 -day OECD 302B Zahn-Wellens test was carried out to assess the inherent biodegradability of HB TMP. The test measured the removal to dissolved organic carbon (DOC) to determine the percentage degradation of the test substance. The DOC was measured after 3 hours and then again on days 3, 7, 14, 21, 27 and 28. The pH was between 6.5 and 8.0 and the temperature was 22.5

±2.5oC throughout the test. Aniline was used as a refence substance to confirm the viability of the activated sludge.

The test substance was found to be 89% after 14 days and 92% after 28 days. As biodegradtion of HB TMP was greater than 70% by day 14, the substance can be considered inherently biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Oct 2019 - 03 Dec 2019

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:

July 1992

Deviations:

yes

Remarks:

Temperature range was 19.1-21.1 degrees celcius, instead of 20.0-24.0. As degradation of the positive control was in the normal range, this was considered as uncritical concerning the outcome of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Slude was taken from a biological sewage treatment plant which predominantly treats domestic sewage. It was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) STP, IM Altenschemel, 67435 NW_Lachen_speyerdorf. It was filtrated, washed with test medium (x2) and re-suspended in the test medium. It was then aerated until use. The dry matter was determined to contain 3.10g of suspended solids/L.

Duration of test (contact time):

28 d

Initial conc.:

39.3 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Pre-Treatment: Aliquots of the test item were made after melting, as described in chapter 6.1.1 under Homogeneity. One of these aliquots of the test item were placed in a sealed bag and immersed in a water bath that was set to a temperature of 80 C until the test item was visibly homogeneous. Then a stock solution containing 1.011 g/L test item in deionised water was prepared. Its organic carbon content was determined in order to estimate the amount to be added to the test flasks. The TOC was 514.27 mg/L, giving an organic carbon content of 50.87 %.
-Instruments and Devices: Data logger for temperature, ebro, Analytical scales Mettler Toledo XS 205 DU, Analytical scales Mettler Toledo XSR205DU, Precision scales Mettler Toledo XS 6001S, Precision scales Sartorius CPA8201, Adjustable pipettes with one-way tips, Mettler Toledo, Automatic repeater pipettes with one-way tips, Mettler Toledo, Carbon analyser TOC multi N/C 2100S, Analytik Jena, Magnetic stirrers, pH-meter 3310, wtw, Heating chamber Heratherm OGS 60, Ultrasonic bath SONOREX RK 510H, Bandelin, Water Bath GFL 1023, Fridge
-Test vessels- 2000 mL-SCHOTT-flasks were used as test vessels, 100 mL scrubber flasks as absorbent vessels.
-Chemicals: All chemicals used in the test were “analytical grade” or with purity >97.5%.
-Positive Control: Aniline (Phenylamine, C6H5NH2, CAS-No. 62-53-3) was used as readily biodegradable positive control. A stock solution containing 2.101 g/L in deionised water was prepared and its
organic carbon content was measured with 1568.3 mg/L, corresponding to an organic carbon content of the positive control of 74.65 %.
-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 conducted 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 (see also chapter 8.3.1). 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.

CO2 DETERMINATION
-Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC
multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon analyser
was calibrated with freshly prepared reference solutions containing potassium hydrogen
phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC). Quality control samples were measured on each measuring day.

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:
𝑒mittC = [(𝐼𝐶(𝑡) − 𝐼𝐶(0)) ∗ 𝑉𝑜𝑙𝑁𝑎𝑂𝐻(𝑡)]/[𝑉𝑜𝑙_𝑇𝑒𝑠𝑡𝑣𝑒𝑠𝑠𝑒𝑙] 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: 0.1 L) - ∑ (all sample volumes up to time t))
Vol_Testvessel test vessel volume in L (here: 1.5)

For day 29, the IC content of both scrubber flasks was taken into account. Calculation of emitted carbon is 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:
% degradation = {[(𝑒𝑚𝑖𝑡𝑡𝑒𝑑𝐶(𝑇𝑒𝑠𝑡)𝑖𝑛 𝑚𝑔⁄𝐿) − (𝑀𝑒𝑎𝑛 𝑒𝑚𝑖𝑡𝑡𝑒𝑑𝐶(𝐶𝑜𝑛𝑡𝑟𝑜𝑙𝑠)𝑖𝑛 𝑚𝑔⁄𝐿)]/(𝑎𝑑𝑑𝑒𝑑𝐶 𝑖𝑛 𝑚𝑔⁄𝐿)}∗ 100%
Degradation in positive control and toxicity flasks was calculated analogously.
Abiotic degradation was calculated from:
% degradation = [(𝑒𝑚𝑖𝑡𝑡𝑒𝑑𝐶(𝑎𝑏𝑖𝑜𝑡𝑖𝑐)𝑖𝑛 𝑚𝑔⁄𝐿)/(𝑎𝑑𝑑𝑒𝑑𝐶 𝑖𝑛 𝑚𝑔⁄𝐿)]∗ 100%

Parameter:

% degradation (CO2 evolution)

Value:

22

Sampling time:

28 d

Remarks on result:

other: no plateau of degradation was reached yet, indicating further biodegradation is possible

Details on results:

The test item HB TMP is considered as “not readily biodegradable“.
-The degree of biodegradation reached 22 % after 28 days.
-The 10-day-window (day 20 – 30) was not detectable, because the end is outside of the test duration.
- Degradation missed 60 % within 28 days, too. 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 HB TMP is considered as
“not readily biodegradable within 28 days”.
- The abiotic degradation was not observed.

Emitted carbon in mg/L

Day	Blank control 1	Blank control 1	Positive control 1	Positive control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
2	0.07	0.25	0.10	0.85	0.35	0.67	0.20	0.22
4	0.54	0.94	1.63	46	0.92	1.46	0.15	2.94
7	1.62	2.17	12.94	12.49	2.51	2.77	0.20	10.44
9	2.06	2.4	14.49	13.35	3.26	3.6	0.11	12.63
11	2.65	2.82	15.97	13.90	3.83	3.95	0.11	14.30
14	3.06	2.99	17.70	15.69	4.53	4.42	0.1	16.14
18	3.6	2.93	19.94	18.47	4.94	4.29	0.07	20.56
23	4.57	3.29	22.57	20.93	6.63	*6.84	-0.01	23.50
29	5.42	5.44	24.34	23.17	8.75	*10.85	-0.17	32.35

* Due to a leakage in the absorber flask of Test 2, a slight loss of NaOH was observed on day 18. Therefore, the measured carbon concentrations after that date were corrected by taking into account the remaining volume in the flask (volume at the end of the test = 70 mL).

Degradation values

Day	Positive control 1	Positive control 2	Positive control mean	test 1	Test 2	Test mean	Abiotic control	Toxicity control
2	-0.3	3.5	1.6	0.9	2.6	1.7	1.0	0.1
4	4.5	19.3	11.9	0.9	3.6	2.3	0.8	5.5
7	55.3	53.1	54.2	3.1	4.4	3.8	1.0	21.4
9	61.4	55.7	58.5	5.2	6.8	6.0	0.6	26
11	66.3	55.9	61.1	5.5	6.1	5.8	0.6	28.9
14	73.5	63.4	68.5	7.5	7.0	7.3	0.5	32.8
18	83.5	76.2	79.9	8.4	5.2	6.8	0.3	43.3
23	93.3	85.2	89.2	13.5	14.6	14.1	0.0	49
29	94.7	88.8	91.8	16.6	27.1	21.9	-0.8	67.4

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:

not readily biodegradable

Conclusions:

As degradation missed the pass level of 60% in the course of the test, HB TMP is considered as not readily biodegradable within 28 days. However, at the end of the test no plateau of degradation was reached yet, indicating further biodegradation is possible.

Executive summary:

In a GLP study according to the OECD 301B (July 1992) testing guideline, HB TMP was evaluated for aerobic elimination adn degradation potential for ready biodegradability using a nominal test item concentration of 20 mg organic carbon/L HB TMP. 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, and all validity criteria were met. Degradation of the positive control surpassed the pass level of 60 % after 10 days. Because the test item is a mixture the 10-day-window does not have to be taken into account. As degradation missed the pass level of 60% in the course of the test, HB TMP is considered as not readily biodegradable within 28 days. However, at the end of the test no plateau of degradation was reached yet, indicating further biodegradation is possible. No observations were made which might cause doubts concerning the validity of the study outcome.

This study is deemed reliable in regard to OECD testing guideline 301B and HB TMP is considered not readily biodegradable.

Description of key information

The test substance was 92% degraded after a 28-day inherent biodegradability test and so can be considered to be inherently biodegradable.

In an OECD 301B testing, HB TMP was not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

A 28 -day OECD 302B Zahn-Wellens test was carried out to assess the inherent biodegradability of reaction mass of 2,2'-[methylenebis(oxymethylene)bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol.

The test measured the removal to dissolved organic carbon (DOC) to determine the percentage degradation of the test substance. The DOC was measured after 3 hours and then again on days 3, 7, 14, 21, 27 and 28. The pH was between 6.5 and 8.0 and the temperature was 22.5 ±2.5oC throughout the test. Aniline was used as a reference substance to confirm the viability of the activated sludge.

The test substance was found to be 89% after 14 days and 92% after 28 days. As biodegradation of the test substance was greater than 70% by day 14, the substance can be considered inherently biodegradable.

In a GLP study according to the OECD 301B (July 1992) testing guideline, HB TMP was evaluated for aerobic elimination adn degradation potential for ready biodegradability using a nominal test item concentration of 20 mg organic carbon/L HB TMP. 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, and all validity criteria were met. Degradation of the positive control surpassed the pass level of 60 % after 10 days. Because the test item is a mixture the 10-day-window does not have to be taken into account. As degradation missed the pass level of 60% in the course of the test, HB TMP is considered as not readily biodegradable within 28 days. However, at the end of the test no plateau of degradation was reached yet, indicating further biodegradation is possible. No observations were made which might cause doubts concerning the validity of the study outcome.

This study is deemed reliable in regard to OECD testing guideline 301B and HB TMP is considered not readily biodegradable.