2-dibutylaminoethanol

Biodegradation in water: screening tests

Description of key information

2-Dibutylaminoethanol is readily biodegradable according to OECD criteria.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

In order to verify the ready biodegradability of 2-dibutyethanolamine (DBEA, CAS 102-81-8) which was already shown in a GLP guideline study according to OECD 301B (BASF SE, 2012, report no. 29G0286/05G008; for details see below), three screening studies were performed in parallel according to OECD TG 301B (enhanced conditions) and under GLP conditions. The studies were performed under the same test conditions but used inocula from three different waste water treatment plants treating predominantly domestic waste water (BASF SE 2019, report no.: 22G0286/05G024, 22G0286/05G025, 22G0286/05G026). The inocula were taken from the following STPs in Germany: Monsheim, Mannheim and Lambsheim. The STP of Mannheim also receives some industrial wastewater in contrast to the other STPs. The studies were set up for 60 d; however, ready biodegradability was evaluated after 28 d.

In the studies using activated sludge from the waste water treatment plants of Mannheim (BASF SE 2019, report no.: 22G0286/05G024) and Lambsheim (BASF SE 2019, report no. 22G0286/05G025), Germany, DBEA was determined to be readily biodegradable. The activated sludge samples used as inocula were not adapted to the substance prior to the study. The 10-day window and the validity criteria were kept in both studies. Using the activated sludge from Mannheim, the degradation reached 83% after 28 d of exposure and 86% after 43 d based on CO2 evolution. Based on DOC removal the final result after 43 d was 103% degradation. In case of the activated sludge from Lambsheim, the degradation reached 76% after 28 d of exposure and 81% after 43 d based on CO2 evolution. Based on DOC removal the final result after 43 d was 93% degradation.

As the validity criteria were kept and the critical level of degradation was achieved within the 10-d window, 2-dibutyethanolamine can be evaluated as readily biodegradable according to OECD criteria. These studies were evaluated as reliable without restriction (RL 1) and selected as key studies.

In the third study which was run in parallel, activated sludge from the waste water treatment plant of Monsheim, Germany, was used (BASF SE 2019, report no.: 22G0286/05G026). The sludge was not adapted to the substance. The degradation reached 23% after 28 d of exposure, but the degradation increased to 93% after prolonged exposure of 57 d based on CO2 evolution. Based on DOC removal the final result after 57 d was 98% degradation. Therefore, DBEA was evaluated as inherently biodegradable under enhanced test conditions. The study was evaluated as reliable without restriction (RL 1) and selected as supporting study.

As described above, a previously performed GLP-guideline study following OECD TG 301B (BASF SE, 2012, report no. 29G0286/05G008), used also activated sludge from a wastewater treatment plant in Mannheim, Germany. The sludge was not adapted to the substance. The 10-day window and the validity criteria were kept. The degradation reached 87% after 28 d of exposure based on CO2 evolution. Based on DOC removal, the substance was degraded almost completely by 98%. The study was evaluated as reliable without restriction (RL 1) and selected as a further key study.

Further screening tests showed that DBEA is biodegradable, but not readily biodegradable under test conditions. The GLP-guideline study according to OECD TG 301A (DOC Die-Away Test) resulted in a degradation of less than 10% based on DOC removal (BASF AG, 2005, report no. 21G0286/053098). The inoculum was activated sludge from a municipal wastewater treatment plant, which was not adapted to the substance. The study was evaluated as reliable without restriction (RL1), as it was performed according to an OECD TG without deficiencies under GLP conditions, and the validity criteria were kept. This study resulted in the conclusion that the substance is not readily biodegradable.

A further ready biodegradability study was performed by NITE (2001, report no. 21539). This is also a GLP-guideline study according to OECD TG 301C (Modified MITI Test (I)). The report was available in Japanese with a result table in English as well as basic information on the test in English via the NITE database. The validity criteria are kept. The inoculum used in this study is a mixture of samples of sludge, surface soil, water, etc. from at least ten sites including sewage treatment works, industrial waste-water treatment works, rivers, lakes, and seas. This mixed inoculum is cultivated in the laboratory prior to use for at least one month and up to four months using an artificial feeding substrate containing glucose, peptone and potassium orthophosphate.

The degradation of DBEA was 1% based on BOD. NITE assessed the substance as non-biodegradable. As only the abstract and a result table was available in English from the reliable NITE database, while detailed information was in Japanese, the study was evaluated as reliable with restrictions (RL 2) as it was performed according to an OECD TG without deficiencies under GLP conditions, and the validity criteria were kept.

According to ECHA Guidance document R.7b (v4.0, June 2017, Ch. R.7.9.4.1; p. 208) positive test results of ready biodegradability tests should generally supersede negative test results realizing that ready biodegradability tests may sometimes fail because of the stringent test conditions. Therefore, DBEA can be concluded to be readily biodegradable according to OECD criteria and in accordance with REACH based on the studies by BASF SE (2012, 2019).

However, as conflicting results are presented, the data are discussed further.

All of the ready biodegradability tests are based on non-adapted inoculum from guideline-conform sources. However, the non-biodegradability in the MITI (I) study might be due to the pre-treatment of the inoculum according to the guideline (laboratory culture of at least 1 month), which may seriously lower the diversity and biodegradation capacity of the microbes (Forney et al., 2001; Kayashima et al., 2014 as cited in the ECHA Guidance document R.7B, p. 210).

In addition, the test substance concentration was very high (100 mg/L test material) in the OECD TG 301C study in compliance with the guideline, which increases the probability of inhibition of the inoculum. Test material concentrations in the two other ready biodegradability tests by BASF (OECD TG 301A, 2005 and 301B, 2012) used 20 mg/L TOC which is equivalent with approximately 30 mg/L test material. The latest ready biodegradability tests used a lesser amount of test material: 15 mg/L (= 10 mg/L DOC),

Although, the short-term respiration inhibition test (OECD TG 209, GLP, BASF AG, 2005, report no. 08G03286/053095) did not indicate inhibition of the microorganisms in municipal activated sludge, due to the different source and cultivation of the inoculum in the MITI test, inhibition of the degradation activity might have occurred.

In conclusion, the OECD TG 301C test is generally to be considered as the most “conservative” ready test compared to the other OECD ready tests due to the above mentioned reasons (high test material concentration, pre-treated inoculum).

In the OECD TG 301A and 301B studies (BASF 2005, 2012), the basic test conditions regarding the concentration of test substance and inoculum are very similar (approximately 30 mg/L test material; inoculum: 30 mg/L dry matter of activated sludge). Inhibition controls and positive controls (= reference substance) do not indicate significant quality differences between the inocula of the two tests. One basic difference in the test conditions is the total test volume used. The tests from 2012 and 2019 by BASF SE (OECD TG 301B) used 1500 mL, while the older study (OECD TG 301A, BASF AG, 2005) used “only” 1000 mL. Although the concentration of the microorganisms is the same, the absolute numbers and the number of different types of microorganisms are different (Ingerslev et al., 2001, cited in REACH Guidance Document R.7b, v4.0, June 2017). In case of the OECD TG 301B study, the higher number of microorganisms must have enhanced the probability of the presence of microbes being able to degrade 2-dibutylaminoethanol.

Overall, it may be concluded that the combination of the different sources of the activated sludge and thus the composition of the microbial population as well as the absolute number of microorganisms per test assay might be the cause for the difference in the substance degradability between the studies.

The information on inherent biodegradability cannot be used to fulfill the standard information requirement of ready biodegradation according to REACH nor can the information be used for classification purposes according to CLP (Regulation (EC) No 1272/2008). According to REACH Guidance Document R.7b (v4.0, June 2017; Ch. 9.4.1; p. 215–216), information of inherent biodegradability tests (OECD TG 302 A–C) can be considered for information on the type of biodegradability. Biodegradation above 70% may be regarded as evidence of inherent, ultimate, biodegradability. Data on inherent biodegradability can be used in the persistence assessment of a substance.

Therefore, the information of the OECD TG 302B study (BASF AG, 1985; report no. 01.1120) are provided in order to support the assessment of 2-dibutylaminoethanol as biodegradable. Since the degree of degradation in the two ready biodegradability studies by BASF AG (2005) and NITE (2001) were very low. The BASF AG (1985) study was assessed as reliable with restrictions as basic data were given. Nevertheless, the documentation of the study falls short of today's requirements, but corresponds to the standards at the time they were conducted; the quality of the study performance is nevertheless not influenced. In the Zahn-Wellens test according to OECD TG 302B using activated sludge from a biological treatment plant receiving primarily industrial wastewater as inoculum, the substance was removed from water to 91% based on DOC in 28 d. After 3 h the measured DOC removal was below 5% indicating that adsorption to activated sludge is not a relevant removal process. It can be concluded that the substance is essentially biodegradable due to the observed lag phase (< 10% DOC-elimination) of approximately 3 d and the remaining course of the degradation curve.

However, as already stated above, the test is not suitable to fulfill the criteria for ready biodegradability according to REACH Annex VII and cannot be used for classification purposes. Therefore, the substance was assessed to be inherently biodegradable (note: inoculum was from an industrial source).

Summing up the available experimental data, the substance is to be considered as readily biodegradable according to OECD criteria. According to ECHA Guidance document R.7B (v4.0, June 2017, Ch. R.7.9.4.1; p. 208), it is justified to select the studies by BASF SE (2012, report no. 29G0286/05G008; 2019, report no. 22G0286/05G024, 22G0286/05G025) as key studies as positive results supersede negative results. The quality of the experimental studies is high (RL1) giving no reason to prefer one over the other study. The negative experimental results can be explained by a combination of several factors, e.g. differences in the inoculum, the test concentration, the test volume.

 

Study / Model	Guideline	GLP	RL[a]	Rationale for RL	Inoculum	Removal in 28 d	Validity criteria fulfilled	Conclusion
Key study: BASF SE (2012), report no. 29G0286/05G008	OECD 301B	Yes	1	GLP guideline study, no deficiencies, validity criteria kept	Activated sludge, domestic, non-adapted	87% (CO2), 98% (DOC)	Yes	Readily biodegradable; 10-d window kept
Key study: BASF SE (2019), report no. 22G0286/05G024	OECD 301B	Yes	1	GLP guideline study, no deficiencies, validity criteria kept	Activated sludge, domestic, non-adapted	83% (CO2), 103% (DOC)[b]	Yes	Readily biodegradable; 10-d window kept
Key study: BASF SE (2019), report no. 22G0286/05G025	OECD 301B	Yes	1	GLP guideline study, no deficiencies, validity criteria kept	Activated sludge, domestic, non-adapted	76% (CO2), 93% (DOC)[b]	Yes	Readily biodegradable; 10-d window kept
Supporting study: BASF SE (2019), report no. 22G0286/05G026	OECD 301B	Yes	1	GLP guideline study, no deficiencies, validity criteria kept	Activated sludge, domestic, non-adapted	23% (CO2), 98% (DOC)[c]	Yes	Inherently biodegradable
Other information: BASF AG (1985), report no. 01.1120	OECD 302B	No	2	Basic data given: comparable to guideline study; sufficient data suitable for assessment.	Activated sludge, industrial	91% (DOC; 1% in 3 h)	No data	Inherently biodegradable
Other information: BASF AG (2005), report no. 21G0286/053098	OECD 301A	Yes	1	GLP guideline study, no deficiencies, validity criteria kept	Activated sludge, domestic, non-adapted	< 10% (DOC)	Yes	Not readily biodegradable
Other information: NITE (2001), report no. 21539	OECD 301C	Yes	2	Basic data given in English, detailed documentation in Japanese; validity criteria kept; reliable database	Mixed inoculum[d]	1% (BOD) 0% (TOC) 3% (test material)	Yes	Not readily biodegradable

[a]RL = reliability

[b]Enhanced test conditions, 43 days (DOC)

[c]Enhanced test conditions, 57 days (DOC), mean of two replicates

[d]Sludge was taken from 10 different sites in Japan (municipal STPs, industrial STPs, lakes, rivers and bays). The sludge is cultured in the laboratory for a period between one and three months using an artificial feeding substrate containing peptone, glucose and potassium orthophosphate.

 

 

Overall conclusion on ready biodegradability

1.    Available experimental data on biodegradability of 2-dibutylaminoethanol (DBEA, CAS 102‑81‑8):

-      Key study: BASF SE (2012): OECD TG 301B, GLP, validity criteria kept, RL 1: Readily biodegradable, 10-d window kept (87% in 28 d; CO2)

-      Key study: BASF SE (2019): OECD TG 301B, GLP, validity criteria kept, RL 1: Readily biodegradable, 10-d window kept (83% in 28 d; CO2), waste water treatment plant Mannheim, Germany

-      Key study: BASF SE (2019): OECD TG 301B, GLP, validity criteria kept, RL 1: Readily biodegradable, 10-d window kept (76% in 28 d; CO2), waste water treatment plant Lambsheim, Germany

-      Supporting study: BASF SE (2019): OECD TG 301B, GLP, validity criteria kept, RL 1: Inherently biodegradable, 10-d window failed (23% in 28 d, CO2; 98% in 57 d, DOC), waste water treatment plant Monsheim, Germany

-      Other information: BASF AG (1985): OECD TG 302B, RL 2: Inherently biodegradable (activated sludge from industrial source; 91% in 28 d, DOC)

-      Other information: BASF AG (1985): OECD TG 301A, GLP, validity criteria kept, RL 1: Not readily biodegradable (< 10% in 28 d, DOC)

-      Other information: NITE (2001): OECD 301C, GLP, validity criteria kept, RL 2: Not readily biodegradable (1% in 28 d, BOD)

2.     Six reliable and valid studies on the ready biodegradability of 2-dibutylaminoethanol are available.

-      DBEA was not readily biodegradable in the conservative OECD TG 301C test by NITE (2001). This test method is regarded as stringent based on the high test material concentration, the reduced microorganism diversity caused by pre-treatment of the sludge and the low test volume).

-      In the test according to OECD 301A, DBEA was also not readily biodegradable (BASF AG, 2005).

-      In one of the OECD TG 301B studies of BASF SE (2019, report no. 22G0286/05G026), DBEA was not readily but inherently biodegradable.

-      In three of the four OECD TG 301B studies of BASF SE (2012; report no. 29G0286/05G008, 2019; report no. 22G0286/05G024, 22G0286/05G025), DBEA fulfilled the criteria for ready biodegradability.

-      It can be concluded that DBEA is readily biodegradable according to OECD criteria considering the guidance on conflicting results provided in section R.7.9.4. of R.7b (v4.0, June 2017) and section 4.1.3.2.4.5. of the Guidance on Application of the CLP criteria (v5.0, July 2017).

-      The differences in the biodegradability of the three applied test methods are probably due to differences in

-      Inoculum (diversity of microorganisms)

-      Test volume (total number of microorganisms and species)

-      Test material concentration (OECD TG 301C: 3x concentration of OECD TG 301A and B)