Propanol, iminobis-, N-(C16-18 and C18-unsatd. alkyl) derivs.

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test performed according slightly modified OECD guidelines wihtout GLP, no validity criteria reported (with exception of endogenous respiration), no certificate of analysis. Biodegradation of tallowamine, propoxylated (2PO) has been assessed in the OECD SCAS test and data found have been combined with results from the OECD closed bottle test.

Justification for type of information:

Biodegradation screening tests performed according OECD 301D and 302A guidelines in order to assess the biodegradability of the test substance and determine the optimal test conditions to be used in a final GLP Closed Bottle test (OECD 301D).

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

see paragraph "study design" and principles of method if other than guideline

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test were introduced; a) ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river water was used as inoculum.

Justfiication of the omission of ammonium chloride from the mineral salts medium of the OECD 301D ready biodegradation test

The ready biodegradation test medium
Ammonium chloride is added to the standard ready biodegradation test (RBT) medium as macro-nutrient for the growth of the microorganisms that are responsible for the biodegradation of the test substance. Ammonium chloride is added in RBT tests under the assumption that there is a nitrogen limitation in RBTs. The OECD 301D test uses however the lowest test substance concentration of all RBTs and the omission of ammonium chloride does not result in nitrogen limitation as shown by the biodegradation of the reference compound in these tests.
Ammonium chloride added with the medium in the OECD 301D test results in an excess of ammonium in the test which will be oxidized to nitrate. Nitrification is performed by a small group of autotrophic bacteria which are not involved in the mineralization of the test substance. The test substance will be mineralized by heterotrophic bacteria. Adding ammonium chloride to the standard ready biodegradation test medium, not needed for growth of the heterotrophic bacteria, results only in growth of the nitrifying autotrophic bacteria.


Stringency of RBTs
A positive RBT result informs about three aspects of biodegradation: i) the presence and pervasiveness of competent microorganisms in the environment; ii) the ultimate biodegradation of the test substance; and iii) rates of biodegradation in the environment. RBTs are regarded as the most stringent biodegradation tests because biodegradation needs to be achieved within a certain time frame using a relatively small inoculum and a relative high concentration of one test substance.
Meaning, in the relatively small RBT inoculum already enough competent microorganisms should be present to achieve the mineralization of the test substance in the required time frame. The inoculum concentration required for ready biodegradation test is described in the OECD guidelines and is measured at the start of an RBT. Hence, the stringency of RBTs is confirmed (checked) at the start of the RBT.
Organic substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing the substance as carbon and energy source. The ammonium present in the standard RBT medium is oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) and are not involved in the biodegradation of organic substances. Hence, the numbers of nitrifying bacteria in the inoculum of RBTs do not affect/influence the stringency of the tests.


Accuracy of RBTs
In RBTs the respiration of the inoculum blank (the endogenous respiration*) is used as the value for the background respiration. On top of this background respiration, it should be possible to accurately measure the respiration by the test substance. In the OECD 301D test there is only a maximum amount of oxygen available at the start of the OECD 301D test (~9 mg O2/L at 20°C, saturation of oxygen in water). Oxygen concentrations in the test should stay aerobic (≥ 0.5 mg O2/L) and a maximum endogenous respiration of 1.5 mg O2/L is allowed. This means that there is ~7 mg O2/L left for the biodegradation assessment of the test substance. If the endogenous respiration would use more oxygen there is less oxygen available to assess the biodegradation of the test substance resulting in a less accurate biodegradation assessment. The validity criteria of the inoculum blank therefore ensures the accuracy of the measured oxygen consumption by the test substance.


* Endogenous respiration is defined as: a situation in which living organisms oxidize some of their own cellular mass instead of oxidizing substrates they take from the environmental matrix.


The influence of the nitrification of the ammonium nitrogen supplemented with the mineral salts medium on the accuracy validity criteria was compared for the different respirometric RBTs. The biodegradation assessment in the OECD 301B test is based on the measurement of evolved CO2 and the accuracy of the test is therefore not affected by the nitrification of the ammonium nitrogen in the mineral salt medium. The OECD 301C, 301D and 301F are respirometric tests based on measurement of the oxygen consumption. Nitrification in the control bottles will in these tests be mistaken for endogenous respiration. Assuming a complete oxidation of the mineral salts ammonium nitrogen results in an additional oxygen consumption of 0.6 mg/L in the OECD 301D test and of 6 mg/L in the OECD 301C and 301F test. The contribution of this oxygen consumption is 40% of the allowed endogenous respiration in the OECD 301D test and is only 10% of the allowed endogenous respiration in the OECD 301C and 301F test. For the OECD 301D test it is known (see first paragraph above) that nitrogen is not limited in the test and therefore the supplemented ammonium nitrogen is an excess that will be oxidized to nitrate. In the OECD 301C and 301F a higher test substance concentration is used and therefore the nitrogen is expected to be limited to biodegrade all the test substance. In these tests part of the dosed mineral salts ammonium nitrogen will be incorporated in new biomass and the actual oxygen consumption by the nitrification is therefore expected to be < 6 mg/L. The contribution of the oxygen consumption of the allowed endogenous respiration in the OECD 301C and 301F will be <10%.

In conclusion: The back-ground respiration of the other RBTs is not or only slightly influenced by the addition of ammonium in the mineral salts medium compared to the influence it has on the back-ground respiration in the OECD 301D test.


High level of variation in RBT results.
The amount and rate of oxygen consumption by the nitrification of the ammonium chloride dosed with the mineral salts medium will mainly depend on the initial numbers of nitrifying bacteria present in the inoculum. These numbers will vary throughout the year because of the seasonal changes. Nitrifying bacteria are sensitive and relative slow growing bacteria. Low bacteria numbers at the start of the tests or an initial delay in growth by toxic effects is therefore not easily to overcome over a 28 days test period. A test substance that is (slightly) toxic to nitrifying bacteria will delay or stop the growth of nitrifying bacteria in the test bottles. In such a case the inoculum blank (with no hampering of the growth of nitrifying bacteria) will overestimate the background respiration in the test bottles resulting in lower (false) biodegradation values. Analysis of formed nitrate and nitrite in the OECD 301D test and control bottles allow a correction for the additional oxygen consumption. These analyses will however also introduce analytical inaccuracy and hence an increased variation (inaccuracy) of the final calculated test substance biodegradation.
The endogenous respiration in the OECD 301D test medium without addition of ammonium chloride and using bacterial densities (cells/L) in the prescribed range of the test guideline varies in general in the range of 1 ± 0.2 mg/L. The addition of ammonium nitrogen in the mineral salts medium would result in max 0.6 mg/L addition oxygen consumption and could therefore result in failing the endogenous respiration validity criteria. The result of adding ammonium chloride in the OECD 301D will therefore result in a higher chance of invalidating test results.

In summary:
An accurate and stringent assessment of the biodegradation potential of a test substance in the OECD 301D test is possible by omitting the ammonium chloride from the RBT test medium. The omission of ammonium chloride from the OECD 301D medium is justified because:
• There is no nitrogen limitation for the growth of heterotrophic micro-organisms in the OECD 301D test and therefore no additional nitrogen source needs to be added with the medium;
• The omission of ammonium chloride does not affect the stringency of the test because the initial bacterial density at the start of the test is demonstrated to be in the prescribed range;
• The omission of ammonium chloride improves the accuracy of the measured oxygen consumption by the test substance.
• It is much more difficult in the OECD 301D test compared to the other RBTs to fulfill the accuracy criteria when ammonium chloride is added (“biased” effect of ammonium between RBTs).
• Supplementing ammonium chloride in the mineral salts medium of the OECD 301D test will introduce a higher variability and more invalidated test results caused by nitrifying bacteria which are not involved in the biodegradation of the test substances.

Based on the above the omission of ammonium chloride from the OECD 301D test medium should be accepted. In addition, test results from OECD 301D tests where ammonium chloride was omitted from the medium are also accepted in other regulations (a.o. biocidal product directive, ecolabeling, etc…).



Justification for the use of river water as test medium:

According to OECD TG 301 it is clearly stated that an alternative source for the inoculum like surface water (e.g. river water) can be used for the test. Furthermore, also the
REACH guidance on Information Requirements and Chemical Safety Assessment (2017) mentions and justifies that micro-organisms (~10^5 cells/mL) in surface waters can be used as inoculum for the closed bottle test.
In principle, organic chemicals should be introduced in all vessles (bottles) of OECD TG 301 and OECD TG 310 irrespective of the inoculum used. The biodegradable organic carbon introduced should in all cases be limited in order to guarantee a low respiration by the microorganisms introduced. Endogenous respiration (oxidation of storage material and protein) by the micro-organisms introduced with inocula is the major controbutor and should be <= 1.5 mg/L at day 28 in the closed bottle test (validity criterion). The endogenous respiration in the blank control bottles (river water) was 1.3 mg/L at day 28. When this validity criterion is met, it is shown that the test substance is the major source of carbon for energy and growth in the test.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: Unadapted river water as well as secondary activated sludge and primay settled sewage

Details on inoculum:

Inoculum and settled sewage
Secondary activated sludge and primary settled sewage were collected from the WWTP Nieuwgraaf in Duiven, The Netherlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic sewage. The primary settled sewage was collected weekly and stored at -20°C until required. 150 ML of secondary activated sludge containing approximately 2 g DW/L of suspended solids was used as an inoculum for each unit.
The activated sludge for the Closed Bottle test was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992). Adapted sludge from the SCAS unit was used upon sampling.


River water was sampled from the Rhine near Heveadorp, The Netherlands. River water was aerated for 5 to 7 days before use. River water
with particles (slightly turbid) and without particles was used as inoculum. The particles were removed by sedimentation.



Justification for the use of river water as test medium:

According to OECD TG 301 it is clearly stated that an alternative source for the inoculum like surface water (e.g. river water) can be used for the test. Furthermore, also the
REACH guidance on Information Requirements and Chemical Safety Assessment (2017) mentions and justifies that micro-organisms (~10^5 cells/mL) in surface waters can be used as inoculum for the closed bottle test.
In principle, organic chemicals should be introduced in all vessles (bottles) of OECD TG 301 and OECD TG 310 irrespective of the inoculum used. The biodegradable organic carbon introduced should in all cases be limited in order to guarantee a low respiration by the microorganisms introduced. Endogenous respiration (oxidation of storage material and protein) by the micro-organisms introduced with inocula is the major controbutor and should be <= 1.5 mg/L at day 28 in the closed bottle test (validity criterion). The endogenous respiration in the blank control bottles (river water) was 1.3 mg/L at day 28. When this validity criterion is met, it is shown that the test substance is the major source of carbon for energy and growth in the test.


Justification for the use of river water as test medium:

OECD tests of the 301 series and OECD 310 are characterized by their low inoculum concentration which mainly determines their stringency. A correct biomass concentration in the OECD 301 series and OECD 310 test is guaranteed by the validity criterium limiting the (endogenous) respiration in the control bottles. The endogenous respiration in the control is therefore a measure of the biomass concentration.
In the OECD 301D test the prescribed biomass concentration is in the range of 10^4 – 10^6 cells/L. The biodegradation of di (2-hydroxyproply) tallow amine (new CAS 1305599-79-0, old CAS 68951-72-4) was tested using the OECD 301D test with particle free river Rhine water (sampled from the river Rhine near Heveadorp, the Netherlands) as inoculum. The river water was aerated for 7 days before use to reduce the endogenous respiration. No information on the actual cell numbers in the inoculum are reported however the endogenous respiration in the test was 1.3 mg/L at day 28 which suggests that the biomass concentration was in the correct range.
Table 1 below shows an overview of measured endogenous respirations and the corresponding inoculum concentrations, determined by colony count method (ISO 6222, 1999), in OECD 301D GLP tests that were performed with particle free river Rhine water. The river water in these studies was sampled from the same place, River Rhine near Heveadorp (the Netherlands), as the GLP study for di (2-hydroxyproply) tallow amine. All river water samples were also aerated for 7 days before use in order to reduce the endogenous respiration. The endogenous respiration from all these river water samples ranged from 0.75 – 1.50 mg/L and the inoculum concentration ranged from 6*10^4 to 1*10^6 CFU/L. These results demonstrate that the river water fulfilled the endogenous respiration validity criterium and contained a biomass concentration that is in the prescribed range of the OECD 301D TG. The measured endogenous respiration in the OECD 301D test with di (2-hydroxyproply) tallow fulfilled the validity criterium (<1.5 mg/L at day 28) and therefore it can be concluded that the test also contained a biomass concentration in the prescribed range of the OECD 301D test guideline.


Table 1 Overview of measured endogenous respirations and the corresponding inoculum concentration in particle free river Rhine water sampled near Heveadorp (the Netherlands) from different OECD TG 301D studies throughout 2020 – 2022 under GLP conditions
OECD 301D GLP study Date sampling river water Endogenous respiration day 28 (mg/L) Inoculum concentration (colony
forming units/L)
T20007 C 02-Mar-2020 1.25 9 * 10^5
T20009 C 30-Mar-2020 0.75 9 * 10^5
T20015 C 11-May-2020 1.50 1 * 10^6
T20018 C 29-Jun-2020 1.45 1 * 10^6
T20019 C 14-Sep-2020 1.05 5 * 10^5
T20023 C 12-Oct-2022 1.05 1 * 10^6
T20026 C 09-Nov-2020 0.95 9 * 10^5
T20027 C 09-Nov-2020 0.95 9 * 10^5
T21001 C 01-Mar-2021 1.00 1 * 10^6
T21004 C 05-Apr-2021 1.45 4 * 10^5
T21015 C 21-May-2021 1.00 4 * 10^5
T21019 C 28-Jun-2021 1.40 5 * 10^5
T22006 C 21-Mar-2022 1.45 1 * 10^5
T22016 C 27-Jun-2022 1.45 6 * 10^4

Duration of test (contact time):

28 - 84 d

Initial conc.:

1 - 2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Closed bottle test

Parameter followed for biodegradation estimation:

TOC removal

Remarks:

SCAS test

Details on study design:

The semi-continuous activated sludge (SCAS) test is the most appropriate inherent biodegradability test available, because it has a greater potential for promoting biodegradation than most other biodegradability tests. Closed Bottle tests inoculated with unadapted and adapted (from SCAS test) activated sludge are suitable to predict the biodegradability of substances which are not in the aqueous phase of biodegradation tests because of their capacity to adsorb and/or poor water solubility. In the SCAS test activated sludge is exposed to a specified concentration of the test substance and the non-purgeable organic carbon (NPOC) is analysed frequently to determine the carbon removal. This test has been performed according to modified OECD Test Guidelines (OECD, 1981).
The SCAS test was performed according to Test Guidelines (OECD, 1981). The test was performed in 150 mL SCAS units. At the start each SCAS unit was filled with 150 mL of activated sludge and the aeration was started. After 23 hours the aeration was stopped and the sludge was allowed to settle for 45 minutes. After settling 100 mL of the supernatant liquor was withdrawn from the tap. Subsequently, 95 mL of primary settled sewage and 5 mL deionized water were added to the control unit, and 95 mL of primary settled sewage and 5 mL of the test compound stock suspension were added to the test unit. Tallowamine, propoxylated (2 PO) was added to the test unit using a stock solution of 1.0 g/L. Aeration was started again and continued for 23 hours. The above fill and draw procedure was repeated 6 times per week throughout the test. Supernatant drawn off was analyzed for non-purgeable organic carbon (NPOC) andxorganic nitrogen content. The NPOC values were used to follow the removal of the test substance. The percentage removal in the SCAS unit was calculated by the following equation:
% removal = 100 (CT-(Ct-Cc)) / CT
CT is the concentration of the test compound as NPOC added to the settled sewage at the start of the aeration period.
Ct is the concentration of NPOC found in the supernatant liquor of the test at the end of the aeration period.
Cc is the concentration of NPOC found in the supernatant liquor of the control.

The Closed Bottle test has been chosen as the most appropriate ready biodegradability test available. The Closed Bottle tests were performed according to slightly modified EU, OECD and ISO Test Guidelines (OECD, 1992). A number of Closed Bottle tests were modified to permit prolonged measurements (van Ginkel and Stroo 1992). The Closed Bottle tests have been inoculated with river water and activated sludge. Furthermore, methods to decrease possible toxic effects of the test substance to the inocula have been carried out to determine the true biodegradability of the test substance (van Ginkel et al, 2008).
Closed Bottle tests.
The Closed Bottle tests were performed according to OECD Test Guidelines (1992). The test was performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers. Use was made of bottles containing only inoculum and bottles containing tallowamine, propoxylated (2 PO) (1.0 or 2.0 mg/L). In some cases, tallowamine, propoxylated (2 PO) was reacted with humic acid and lignosulphonic acid, respectively, both at a concentration of 2.0 mg/L in the bottles.Possible toxic effects of tallowamine, propoxylated (2 PO) were also counteracted through addition of 2 g or 4 g of silica gel in the bottles. During the test period, the test substance should be released slowly from the silica gel. Although no additional oxygen consumption was expected, controls with silica gel, humic acid and lignosulphonic acid were carried out as well. The inoculum was diluted to 2 mg DW/L in the closed bottles. The clear river water was used undiluted. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated at 23±1°C in the dark. The biodegradation was measured by following the course of the oxygen decrease in the bottles with a special funnel. This funnel fitted exactly in the BOD bottle (van Ginkel and Stroo 1992).

Test performance:

The ThODs of the active ingredient, tallowamine, propoxylated (2 PO) is 2.4 mg/mg. The chemical oxygen demand of tallowamine, propoxylated (2 PO) is 2.2 mg/mg. The ThOD
has been used to calculate the biodegradation percentages. The pH of the medium was 7.0 at the start of the test. The pH of the medium with sludge at day 28 was 6.8 to 7.2. The
pH in the bottles with river water was 7.8 to 8.1. Temperatures ranged from 22 to 24°C. The validity of the tests is demonstrated by oxygen concentrations >0.5 mg/L in all bottles
during the test period.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

closed bottle test based on ThOD-NO3

Value:

67

Sampling time:

28 d

Remarks on result:

other: Readily biodegradable based on measured test results in the presence of silica gel by using unadapted river water

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

closed bottle test based on ThOD-NH3

Value:

71

Sampling time:

28 d

Remarks on result:

other: Readily biodegradable based on measured test results in the presence of silica gel by using unadapted river water

Parameter:

% degradation (O2 consumption)

Value:

63

Sampling time:

28 d

Remarks on result:

other: adapted activated sludge from scas test

Parameter:

% degradation (O2 consumption)

Value:

66

Sampling time:

84 d

Remarks on result:

other: unadapted sludge

Parameter:

% degradation (TOC removal)

Remarks:

SCAS test

Value:

90 - 100

Sampling time:

100 d

Remarks on result:

other: 60% TOC removal from the start

Details on results:

ThOD based on average molecular formula calculated (C24H52O2N) and assuming a 100% organic purity
ThODNH3 = 2.92 mgO2/mg; ThODNO3 = 3.08 mgO2/mg


71% biodegradation CBT (based on ThOD-NH3) and 67% biodegradation (based on ThOD-NO3): 1 mg/L of tallowamine, propoxylated (2PO) was tested in the closed bottle test in the presense of silica gel. Unadapted river water (clear, undiluted) was used as medium/inoculumn.

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles with 1 mg/L test substance and the addition of silica gel in unadapted river water.

 

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot
0	8.8	8.8
	8.8	8.8
	8.8	8.8
Mean (M)	8.8	8.8
7	8.3	7.7
	8.2	7.7
	8.3	8.0
Mean (M)	8.3	7.8
14	7.8	6.6
	8.0	6.8
	8.0	6.4
Mean	7.9	6.6
21	7.7	5.7
	7.5	5.8
	7.5	5.9
Mean (M)	7.6	5.8
28	7.5	5.5
	7.4	5.4
	7.5	5.3
Mean (M)	7.5	5.4

Ocs Mineral nutrient solution without test material but with inoculum and silica gel.

Ot Mineral nutrient solution with test material (1.0 mg/L), silica gel, and inoculum.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tallowamine, propoxylated (2 PO) is not readily biodegradable in a “standard” test. The inhibition of the endogenous respiration clearly demonstrates that biodegradation is

jeopardized by the toxicity of the test substance . Inhibition of biodegradation by the test substance can in some cases be prevented through the addition of humic acid,

lignosulphonic acid or silica gel, all known for their capacity to reduce the toxicity of cationic surfactants (van Ginkel et al, 2008). The best result was obtained with silica gel.

Inhibition of the test substances was not or only slightly counteracted by lignosulphonates and humic acid. The highest chance of finding ready biodegradability

is with river water and silica gel. A biodegradation in excess of 60% is easily

achieved in a prolonged Closed Bottle test (enhanced biodegradability test). This result has been shown to be reproducible.

For a final GLP test it is recommended to use river water without particles as inoculum and silica gel to prevent toxicity.

Validity criteria fulfilled:

not specified

Remarks:

Performed under standard conditions which accertain valid condition. The validity of the test is demonstrated by an endogenous respiration of 1.3 mg/L at day 28 for unadapted river water.

Interpretation of results:

readily biodegradable

Conclusions:

The ready biodegradability of di (2-hydroxypropyl)tallow amine has been shown to be reproducible under standard OECD test conditions. The result is despite the absence of GLP and without evaluation of all validity criteria considered to be reliable. The endogenous respiration at day 28 for unadapted river water was 1.3 mg/L.
The ready result was obtained with unadapted inoculum and is therefore adequate for risk assessment purposes according to the REACH legislation.

Executive summary:

Complete biodegradation of tallowamine, propoxylated (2 PO) has been demonstrated with the SCAS results and also in a closed bottle test with unadapted river water and the addition of silica gel biodegradation >60% after 28 days was shown.

It can be concluded that tallowamine, propoxylated (2 PO) is NOT persistent and readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-09 - 2009-10-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

acceptable deviations, please see principles of method if other than giuideline.

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test were introduced; a) ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river water was used as inoculum.


Justfiication of the omission of ammonium chloride from the mineral salts medium of the OECD 301D ready biodegradation test

The ready biodegradation test medium
Ammonium chloride is added to the standard ready biodegradation test (RBT) medium as macro-nutrient for the growth of the microorganisms that are responsible for the biodegradation of the test substance. Ammonium chloride is added in RBT tests under the assumption that there is a nitrogen limitation in RBTs. The OECD 301D test uses however the lowest test substance concentration of all RBTs and the omission of ammonium chloride does not result in nitrogen limitation as shown by the biodegradation of the reference compound in these tests.
Ammonium chloride added with the medium in the OECD 301D test results in an excess of ammonium in the test which will be oxidized to nitrate. Nitrification is performed by a small group of autotrophic bacteria which are not involved in the mineralization of the test substance. The test substance will be mineralized by heterotrophic bacteria. Adding ammonium chloride to the standard ready biodegradation test medium, not needed for growth of the heterotrophic bacteria, results only in growth of the nitrifying autotrophic bacteria.


Stringency of RBTs
A positive RBT result informs about three aspects of biodegradation: i) the presence and pervasiveness of competent microorganisms in the environment; ii) the ultimate biodegradation of the test substance; and iii) rates of biodegradation in the environment. RBTs are regarded as the most stringent biodegradation tests because biodegradation needs to be achieved within a certain time frame using a relatively small inoculum and a relative high concentration of one test substance.
Meaning, in the relatively small RBT inoculum already enough competent microorganisms should be present to achieve the mineralization of the test substance in the required time frame. The inoculum concentration required for ready biodegradation test is described in the OECD guidelines and is measured at the start of an RBT. Hence, the stringency of RBTs is confirmed (checked) at the start of the RBT.
Organic substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing the substance as carbon and energy source. The ammonium present in the standard RBT medium is oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) and are not involved in the biodegradation of organic substances. Hence, the numbers of nitrifying bacteria in the inoculum of RBTs do not affect/influence the stringency of the tests.


Accuracy of RBTs
In RBTs the respiration of the inoculum blank (the endogenous respiration*) is used as the value for the background respiration. On top of this background respiration, it should be possible to accurately measure the respiration by the test substance. In the OECD 301D test there is only a maximum amount of oxygen available at the start of the OECD 301D test (~9 mg O2/L at 20°C, saturation of oxygen in water). Oxygen concentrations in the test should stay aerobic (≥ 0.5 mg O2/L) and a maximum endogenous respiration of 1.5 mg O2/L is allowed. This means that there is ~7 mg O2/L left for the biodegradation assessment of the test substance. If the endogenous respiration would use more oxygen there is less oxygen available to assess the biodegradation of the test substance resulting in a less accurate biodegradation assessment. The validity criteria of the inoculum blank therefore ensures the accuracy of the measured oxygen consumption by the test substance.


* Endogenous respiration is defined as: a situation in which living organisms oxidize some of their own cellular mass instead of oxidizing substrates they take from the environmental matrix.


The influence of the nitrification of the ammonium nitrogen supplemented with the mineral salts medium on the accuracy validity criteria was compared for the different respirometric RBTs. The biodegradation assessment in the OECD 301B test is based on the measurement of evolved CO2 and the accuracy of the test is therefore not affected by the nitrification of the ammonium nitrogen in the mineral salt medium. The OECD 301C, 301D and 301F are respirometric tests based on measurement of the oxygen consumption. Nitrification in the control bottles will in these tests be mistaken for endogenous respiration. Assuming a complete oxidation of the mineral salts ammonium nitrogen results in an additional oxygen consumption of 0.6 mg/L in the OECD 301D test and of 6 mg/L in the OECD 301C and 301F test. The contribution of this oxygen consumption is 40% of the allowed endogenous respiration in the OECD 301D test and is only 10% of the allowed endogenous respiration in the OECD 301C and 301F test. For the OECD 301D test it is known (see first paragraph above) that nitrogen is not limited in the test and therefore the supplemented ammonium nitrogen is an excess that will be oxidized to nitrate. In the OECD 301C and 301F a higher test substance concentration is used and therefore the nitrogen is expected to be limited to biodegrade all the test substance. In these tests part of the dosed mineral salts ammonium nitrogen will be incorporated in new biomass and the actual oxygen consumption by the nitrification is therefore expected to be < 6 mg/L. The contribution of the oxygen consumption of the allowed endogenous respiration in the OECD 301C and 301F will be <10%.

In conclusion: The back-ground respiration of the other RBTs is not or only slightly influenced by the addition of ammonium in the mineral salts medium compared to the influence it has on the back-ground respiration in the OECD 301D test.


High level of variation in RBT results.
The amount and rate of oxygen consumption by the nitrification of the ammonium chloride dosed with the mineral salts medium will mainly depend on the initial numbers of nitrifying bacteria present in the inoculum. These numbers will vary throughout the year because of the seasonal changes. Nitrifying bacteria are sensitive and relative slow growing bacteria. Low bacteria numbers at the start of the tests or an initial delay in growth by toxic effects is therefore not easily to overcome over a 28 days test period. A test substance that is (slightly) toxic to nitrifying bacteria will delay or stop the growth of nitrifying bacteria in the test bottles. In such a case the inoculum blank (with no hampering of the growth of nitrifying bacteria) will overestimate the background respiration in the test bottles resulting in lower (false) biodegradation values. Analysis of formed nitrate and nitrite in the OECD 301D test and control bottles allow a correction for the additional oxygen consumption. These analyses will however also introduce analytical inaccuracy and hence an increased variation (inaccuracy) of the final calculated test substance biodegradation.
The endogenous respiration in the OECD 301D test medium without addition of ammonium chloride and using bacterial densities (cells/L) in the prescribed range of the test guideline varies in general in the range of 1 ± 0.2 mg/L. The addition of ammonium nitrogen in the mineral salts medium would result in max 0.6 mg/L addition oxygen consumption and could therefore result in failing the endogenous respiration validity criteria. The result of adding ammonium chloride in the OECD 301D will therefore result in a higher chance of invalidating test results.

In summary:
An accurate and stringent assessment of the biodegradation potential of a test substance in the OECD 301D test is possible by omitting the ammonium chloride from the RBT test medium. The omission of ammonium chloride from the OECD 301D medium is justified because:
• There is no nitrogen limitation for the growth of heterotrophic micro-organisms in the OECD 301D test and therefore no additional nitrogen source needs to be added with the medium;
• The omission of ammonium chloride does not affect the stringency of the test because the initial bacterial density at the start of the test is demonstrated to be in the prescribed range;
• The omission of ammonium chloride improves the accuracy of the measured oxygen consumption by the test substance.
• It is much more difficult in the OECD 301D test compared to the other RBTs to fulfill the accuracy criteria when ammonium chloride is added (“biased” effect of ammonium between RBTs).
• Supplementing ammonium chloride in the mineral salts medium of the OECD 301D test will introduce a higher variability and more invalidated test results caused by nitrifying bacteria which are not involved in the biodegradation of the test substances.

Based on the above the omission of ammonium chloride from the OECD 301D test medium should be accepted. In addition, test results from OECD 301D tests where ammonium chloride was omitted from the medium are also accepted in other regulations (a.o. biocidal product directive, ecolabeling, etc…).




Justification for the use of river water as test medium:

According to OECD TG 301 it is clearly stated that an alternative source for the inoculum like surface water (e.g. river water) can be used for the test. Furthermore, also the
REACH guidance on Information Requirements and Chemical Safety Assessment (2017) mentions and justifies that micro-organisms (~10^5 cells/mL) in surface waters can be used as inoculum for the closed bottle test.
In principle, organic chemicals should be introduced in all vessles (bottles) of OECD TG 301 and OECD TG 310 irrespective of the inoculum used. The biodegradable organic carbon introduced should in all cases be limited in order to guarantee a low respiration by the microorganisms introduced. Endogenous respiration (oxidation of storage material and protein) by the micro-organisms introduced with inocula is the major controbutor and should be <= 1.5 mg/L at day 28 in the closed bottle test (validity criterion). The endogenous respiration in the blank control bottles (river water) was 1.1 mg/L at day 28. When this validity criterion is met, it is shown that the test substance is the major source of carbon for energy and growth in the test.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Remarks:

river water was used undiluted, spiked with nutrients (see study design) and was the inoculum and medium in the closed bottle test

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands (09-09-2009). The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation.



Justification for the use of river water as test medium:

According to OECD TG 301 it is clearly stated that an alternative source for the inoculum like surface water (e.g. river water) can be used for the test. Furthermore, also the
REACH guidance on Information Requirements and Chemical Safety Assessment (2017) mentions and justifies that micro-organisms (~10^5 cells/mL) in surface waters can be used as inoculum for the closed bottle test.
In principle, organic chemicals should be introduced in all vessles (bottles) of OECD TG 301 and OECD TG 310 irrespective of the inoculum used. The biodegradable organic carbon introduced should in all cases be limited in order to guarantee a low respiration by the microorganisms introduced. Endogenous respiration (oxidation of storage material and protein) by the micro-organisms introduced with inocula is the major controbutor and should be <= 1.5 mg/L at day 28 in the closed bottle test (validity criterion). The endogenous respiration in the blank control bottles (river water) was 1.1 mg/L at day 28. When this validity criterion is met, it is shown that the test substance is the major source of carbon for energy and growth in the test.





Justification for data on the inoculum concentration: 

OECD tests of the 301 series and OECD 310 are characterized by their low inoculum concentration which mainly determines their stringency. A correct biomass concentration in the OECD 301 series and OECD 310 test is guaranteed by the validity criterium limiting the (endogenous) respiration in the control bottles. The endogenous respiration in the control is therefore a measure of the biomass concentration.
In the OECD 301D test the prescribed biomass concentration is in the range of 10^4 – 10^6 cells/L. The biodegradation of di (2-hydroxyproply) tallow amine (new CAS 1305599-79-0, old CAS 68951-72-4) was tested using the OECD 301D test with particle free river Rhine water (sampled from the river Rhine near Heveadorp, the Netherlands on 09-09-2009) as inoculum. The river water was aerated for 7 days before use to reduce the endogenous respiration. No information on the actual cell numbers in the inoculum are reported however the endogenous respiration in the test was 1.1 mg/L at day 28 which suggests that the biomass concentration was in the correct range.
Table 1 below shows an overview of measured endogenous respirations and the corresponding inoculum concentrations, determined by colony count method (ISO 6222, 1999), in OECD 301D GLP tests that were performed with particle free river Rhine water. The river water in these studies was sampled from the same place, River Rhine near Heveadorp (the Netherlands), as the GLP study for di (2-hydroxyproply) tallow amine. All river water samples were also aerated for 7 days before use in order to reduce the endogenous respiration. The endogenous respiration from all these river water samples ranged from 0.75 – 1.50 mg/L and the inoculum concentration ranged from 6*10^4 to 1*10^6 CFU/L. These results demonstrate that the river water fulfilled the endogenous respiration validity criterium and contained a biomass concentration that is in the prescribed range of the OECD 301D TG. The measured endogenous respiration in the OECD 301D test with di (2-hydroxyproply) tallow fulfilled the validity criterium (<1.5 mg/L at day 28) and therefore it can be concluded that the test also contained a biomass concentration in the prescribed range of the OECD 301D test guideline.


Table 1 Overview of measured endogenous respirations and the corresponding inoculum concentration in particle free river Rhine water sampled near Heveadorp (the Netherlands) from different OECD TG 301D studies throughout 2020 – 2022 under GLP conditions
OECD 301D GLP study Date sampling river water Endogenous respiration day 28 (mg/L) Inoculum concentration (colony
forming units/L)
T20007 C 02-Mar-2020 1.25 9 * 10^5
T20009 C 30-Mar-2020 0.75 9 * 10^5
T20015 C 11-May-2020 1.50 1 * 10^6
T20018 C 29-Jun-2020 1.45 1 * 10^6
T20019 C 14-Sep-2020 1.05 5 * 10^5
T20023 C 12-Oct-2022 1.05 1 * 10^6
T20026 C 09-Nov-2020 0.95 9 * 10^5
T20027 C 09-Nov-2020 0.95 9 * 10^5
T21001 C 01-Mar-2021 1.00 1 * 10^6
T21004 C 05-Apr-2021 1.45 4 * 10^5
T21015 C 21-May-2021 1.00 4 * 10^5
T21019 C 28-Jun-2021 1.40 5 * 10^5
T22006 C 21-Mar-2022 1.45 1 * 10^5
T22016 C 27-Jun-2022 1.45 6 * 10^4

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stock solutions
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4·2H2O, 22.5 mg
MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
Sodium acetate and the testsubstance were added to the bottles using stock solutions of 1.0 g/L.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994).
Use was made of 10 bottles containing only river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle),
10 bottles containing river water, silica gel and test substance, and 6 bottles containing sodium acetate and river water. The concentrations
of the test substance and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the
respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed
for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark.
Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Calculation of the results

Calculation of endogenous respiration
the endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
Mc is the mean oxygen level in the control bottle with river water.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of di (2-hydroxypropyl) tallow amine and sodium acetate were calculated from their molecular formulae and molecular weights.
The calculated theoretical oxygen demand (ThOD) of di (2-hydroxypropyl) tallow amine is 2.9 mg/mg. It is assumed that the impurities (2.7%)
and the test substance have comparable ThODs. The ThOD of sodium acetate is 0.8 mg/mg

Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference
compound bottles were calculated as follows:
Oxygen consumptionn (mg/L) by test substance = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or cs is the mean oxygen level in the control bottles filled with river water spiked with mineral salts with and without silica gel n days after the start of the test. Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a),
present in river water spiked with mineral salts n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test compound and sodium acetate was calculated by dividing the oxygen consumption
by the concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Based on ThOD-NO3

Value:

59

Sampling time:

28 d

Remarks on result:

other: Readily biodegradable based on measured test results in the presence of silica gel

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Based on ThOD-NH3

Value:

63

Sampling time:

28 d

Remarks on result:

other: Readily biodegradable based on measured test results in the presence of silica gel

Details on results:

Biodegradation calculated using:
2 mg/L test material
ThOD based on average molecular formula calculated from info CoA (C23,3H47,64O2N) and assuming a 100% organic purity
ThODNH3 = 2.86 mgO2/mg ; ThODNO3 = 3.04 mgO2/mg in the bottles: ThODNH3 = 5.72 mg/L and ThODNO3 = 6.08 mg/L


Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of di (2-hydroxypropyl) tallow amine to microorganisms degrading acetate is not relevant. The test substance was expected to be toxic tomicroorganisms in the Closed Bottle test. The test substance was therefore tested in the presence of silica gel.
Inhibition of the endogenous respiration of the inoculum by the test substance tested in the presence of silica gel was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of di (2-hydroxypropyl) tallow amine is expected.

Test conditions
The pH of the media was 7.9 at the start of the test. The pH of the medium at day 28 was 7.8 (control, control with silica gel and test).
Temperatures were within the prescribed temperature range of 22 to 24°C.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 1.1 mg/L
at day 28 (Table I). Furthermore, the differences of the replicate values at day 28
were less than 20%. The biodegradation percentage of the reference compound,
sodium acetate, at day 14 was 85. Finally, the validity of the test is shown by
oxygen concentrations >0.5 mg/L in all bottles during the test period.

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot	Oc	Oa
0	8.8	8.8	8.8	8.8
	8.8	8.8	8.8	8.8
Mean (M)	8.8	8.8	8.8	8.8
7	8.4	7.8	8.3	4.3
	8.2	7.9	8.4	3.9
Mean (M)	8.3	7.9	8.4	4.1
14	7.9	6.4	8.0	3.3
	8.0	6.6	8.0	3.5
Mean	8.0	6.5	8.0	3.4
21	8.0	5.9	8.0
	7.8	5.7	8.0
Mean (M)	7.9	5.8	8.0
28	7.5	3.8	7.7
	7.5	4.0	7.6
Mean (M)	7.5	3.9	7.7

Ocs Mineral nutrient solution without test material but with inoculum and silica gel.

Ot Mineral nutrient solution with test material (2.0 mg/L), silica gel, and inoculum.

Oc Mineral nutrient solution with only inoculum.

Oa Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum.

Validity criteria fulfilled:

yes

Remarks:

The validity of the test is demonstrated by an endogenous respiration of 1.1 mg/L at day 28. The difference of the replicate values at day 28 were less than 20%. Biodg. percentage of ref. compound was 85 at day 14.

Interpretation of results:

readily biodegradable

Conclusions:

Di (2-hydroxypropyl) tallow amine is biodegraded 62% (based on ThODNH3) and 58% (based on ThODNO3) at day 28 in the Closed Bottle test. The 10-day time window criterion was developed on the assumption that compounds are degraded according to “standard” logarithmic growth curves as found with
water soluble compounds. The 10-day window was not used as criterion because of the low bioavailability and the structure of the substance. Di (2-hydroxypropyl) tallow amine was adsorbed onto silica gel to reduce the toxicity of the test substance
in bottles. The poor availability due to slow release from the silica gel might prevent logarithmic growth.
Surfactants are chemicals in which a hydrophilic group is linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two microorganisms as a single organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). Biodegradation of for instance the hydrophilic part of di (2-hydroxypropyl) tallow amine can not begin until significant amounts of this product are formed by alkyl chain degrading microorganisms.
In ready biodegradability tests, the two moieties of di (2-hydroxypropyl) tallow amine are expected to degrade sequentially. The biodegradation of the two moieties may be fully in line with the 10-day window criterion when judged as separate chemicals.

Test performed under GLP, according the guidelines with minor acceptable deviations, meeting all validity criteria.

Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. Di (2-hydroxypropyl) tallow amine in the presence of silica gel did not cause a reduction in the endogenous respiration. In the presence of silica gel, the test substance is therefore considered to be non-inhibitory to the inoculum. Di (2-hydroxypropyl) tallow amine was biodegraded 62% (based on ThODNH3) and 58% (based on ThODNO3) at day 28 in the Closed Bottle test. The time-window was ignored as a pass fail criterion because of the biodegradation kinetics. Hence this substance should be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.1 mg/L at day 28. Furthermore the differences of the replicate values at day 28 were less than 20% and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 85% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

 

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2022 to February 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)

Version / remarks:

1994

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1992

Deviations:

yes

Remarks:

One minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced: Ammonium chloride was omitted from the medium more details see principles other than guideline

Principles of method if other than guideline:

A minor deviation from the guidelines of the Closed Bottle test (OECD TG 301D) was introduced.
Ammonium chloride present in the mineral salt medium was omitted because it may be
converted to nitrate which leads to an oxygen consumption not related to the biodegradation
of the test substance. The omission of ammonium does not affect the biodegradability because
ammonium is not limiting in the Closed Bottle test as was demonstrated by the biodegradation
of the reference substance. The omission of ammonium does make the quantification of the
biodegradation of the test substance more accurate.



Justfiication of the omission of ammonium chloride from the mineral salts medium of the OECD 301D ready biodegradation test

The ready biodegradation test medium
Ammonium chloride is added to the standard ready biodegradation test (RBT) medium as macro-nutrient for the growth of the microorganisms that are responsible for the biodegradation of the test substance. Ammonium chloride is added in RBT tests under the assumption that there is a nitrogen limitation in RBTs. The OECD 301D test uses however the lowest test substance concentration of all RBTs and the omission of ammonium chloride does not result in nitrogen limitation as shown by the biodegradation of the reference compound in these tests.
Ammonium chloride added with the medium in the OECD 301D test results in an excess of ammonium in the test which will be oxidized to nitrate. Nitrification is performed by a small group of autotrophic bacteria which are not involved in the mineralization of the test substance. The test substance will be mineralized by heterotrophic bacteria. Adding ammonium chloride to the standard ready biodegradation test medium, not needed for growth of the heterotrophic bacteria, results only in growth of the nitrifying autotrophic bacteria.


Stringency of RBTs
A positive RBT result informs about three aspects of biodegradation: i) the presence and pervasiveness of competent microorganisms in the environment; ii) the ultimate biodegradation of the test substance; and iii) rates of biodegradation in the environment. RBTs are regarded as the most stringent biodegradation tests because biodegradation needs to be achieved within a certain time frame using a relatively small inoculum and a relative high concentration of one test substance.
Meaning, in the relatively small RBT inoculum already enough competent microorganisms should be present to achieve the mineralization of the test substance in the required time frame. The inoculum concentration required for ready biodegradation test is described in the OECD guidelines and is measured at the start of an RBT. Hence, the stringency of RBTs is confirmed (checked) at the start of the RBT.
Organic substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing the substance as carbon and energy source. The ammonium present in the standard RBT medium is oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) and are not involved in the biodegradation of organic substances. Hence, the numbers of nitrifying bacteria in the inoculum of RBTs do not affect/influence the stringency of the tests.


Accuracy of RBTs
In RBTs the respiration of the inoculum blank (the endogenous respiration*) is used as the value for the background respiration. On top of this background respiration, it should be possible to accurately measure the respiration by the test substance. In the OECD 301D test there is only a maximum amount of oxygen available at the start of the OECD 301D test (~9 mg O2/L at 20°C, saturation of oxygen in water). Oxygen concentrations in the test should stay aerobic (≥ 0.5 mg O2/L) and a maximum endogenous respiration of 1.5 mg O2/L is allowed. This means that there is ~7 mg O2/L left for the biodegradation assessment of the test substance. If the endogenous respiration would use more oxygen there is less oxygen available to assess the biodegradation of the test substance resulting in a less accurate biodegradation assessment. The validity criteria of the inoculum blank therefore ensures the accuracy of the measured oxygen consumption by the test substance.


* Endogenous respiration is defined as: a situation in which living organisms oxidize some of their own cellular mass instead of oxidizing substrates they take from the environmental matrix.


The influence of the nitrification of the ammonium nitrogen supplemented with the mineral salts medium on the accuracy validity criteria was compared for the different respirometric RBTs. The biodegradation assessment in the OECD 301B test is based on the measurement of evolved CO2 and the accuracy of the test is therefore not affected by the nitrification of the ammonium nitrogen in the mineral salt medium. The OECD 301C, 301D and 301F are respirometric tests based on measurement of the oxygen consumption. Nitrification in the control bottles will in these tests be mistaken for endogenous respiration. Assuming a complete oxidation of the mineral salts ammonium nitrogen results in an additional oxygen consumption of 0.6 mg/L in the OECD 301D test and of 6 mg/L in the OECD 301C and 301F test. The contribution of this oxygen consumption is 40% of the allowed endogenous respiration in the OECD 301D test and is only 10% of the allowed endogenous respiration in the OECD 301C and 301F test. For the OECD 301D test it is known (see first paragraph above) that nitrogen is not limited in the test and therefore the supplemented ammonium nitrogen is an excess that will be oxidized to nitrate. In the OECD 301C and 301F a higher test substance concentration is used and therefore the nitrogen is expected to be limited to biodegrade all the test substance. In these tests part of the dosed mineral salts ammonium nitrogen will be incorporated in new biomass and the actual oxygen consumption by the nitrification is therefore expected to be < 6 mg/L. The contribution of the oxygen consumption of the allowed endogenous respiration in the OECD 301C and 301F will be <10%.

In conclusion: The back-ground respiration of the other RBTs is not or only slightly influenced by the addition of ammonium in the mineral salts medium compared to the influence it has on the back-ground respiration in the OECD 301D test.


High level of variation in RBT results.
The amount and rate of oxygen consumption by the nitrification of the ammonium chloride dosed with the mineral salts medium will mainly depend on the initial numbers of nitrifying bacteria present in the inoculum. These numbers will vary throughout the year because of the seasonal changes. Nitrifying bacteria are sensitive and relative slow growing bacteria. Low bacteria numbers at the start of the tests or an initial delay in growth by toxic effects is therefore not easily to overcome over a 28 days test period. A test substance that is (slightly) toxic to nitrifying bacteria will delay or stop the growth of nitrifying bacteria in the test bottles. In such a case the inoculum blank (with no hampering of the growth of nitrifying bacteria) will overestimate the background respiration in the test bottles resulting in lower (false) biodegradation values. Analysis of formed nitrate and nitrite in the OECD 301D test and control bottles allow a correction for the additional oxygen consumption. These analyses will however also introduce analytical inaccuracy and hence an increased variation (inaccuracy) of the final calculated test substance biodegradation.
The endogenous respiration in the OECD 301D test medium without addition of ammonium chloride and using bacterial densities (cells/L) in the prescribed range of the test guideline varies in general in the range of 1 ± 0.2 mg/L. The addition of ammonium nitrogen in the mineral salts medium would result in max 0.6 mg/L addition oxygen consumption and could therefore result in failing the endogenous respiration validity criteria. The result of adding ammonium chloride in the OECD 301D will therefore result in a higher chance of invalidating test results.

In summary:
An accurate and stringent assessment of the biodegradation potential of a test substance in the OECD 301D test is possible by omitting the ammonium chloride from the RBT test medium. The omission of ammonium chloride from the OECD 301D medium is justified because:
• There is no nitrogen limitation for the growth of heterotrophic micro-organisms in the OECD 301D test and therefore no additional nitrogen source needs to be added with the medium;
• The omission of ammonium chloride does not affect the stringency of the test because the initial bacterial density at the start of the test is demonstrated to be in the prescribed range;
• The omission of ammonium chloride improves the accuracy of the measured oxygen consumption by the test substance.
• It is much more difficult in the OECD 301D test compared to the other RBTs to fulfill the accuracy criteria when ammonium chloride is added (“biased” effect of ammonium between RBTs).
• Supplementing ammonium chloride in the mineral salts medium of the OECD 301D test will introduce a higher variability and more invalidated test results caused by nitrifying bacteria which are not involved in the biodegradation of the test substances.

Based on the above the omission of ammonium chloride from the OECD 301D test medium should be accepted. In addition, test results from OECD 301D tests where ammonium chloride was omitted from the medium are also accepted in other regulations (a.o. biocidal product directive, ecolabeling, etc…).

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on inoculum:

Inoculum
Secondary activated sludge was obtained (17-Oct-2022) from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 μm cellulose nitrate filter. This filter was dried for 1.5 hour at 102.0 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.9 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates.
To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week at ambient temperature. The preconditioned sludge was homogenized before use by pressing it through a sterile needle with a syringe. The homogenized sludge was subsequently diluted further to 2 mg dry weight/L concentration in the bottles (van Ginkel and Stroo, 1992).

The pre-exposure of the activated sludge inoculum to the test substance and the recording of this was not performed in compliance with the GLP standards.

The (non-GLP) method used for the pre-exposure of the activated sludge was as follows: The volume of the SCAS unit was 150-mL. The SCAS unit was filled with 150 mL of activated sludge and the aeration was started. After 23 hours the aeration was stopped, and the sludge was allowed to settle for one hour. After settling 100 mL of the supernatant liquor was withdrawn from the tap of the unit. Subsequently, 100 mL of primary settled sewage was added, and the aeration was started. This fill and draw cycle were repeated daily for one week. After one week 100 mL of the settled supernatant liquor was withdrawn from the tap and subsequent 95 mL of primary settled sewage and 5 mL of test substance stock (2 g/L) was administrated to the SCAS unit which resulted in a test substance concentration of 100 mg/L in de SCAS unit. Aeration was started again and the above fill and draw procedure for dosing the test substance was repeated five times per week throughout the 20 weeks pre-exposure period.

Activated sludge pre-exposed to test substance was sampled for use in the GLP test on 24-Oct-2022 from a semi-continuously-fed activated sludge (SCAS) unit which was operated according to the OECD 302A Test Guideline (OECD, 1981). The pre-exposed activated sludge was sampled at the end of the SCAS cycle after 20 weeks of pre-exposure. The dry weight of the pre-exposed activated sludge inoculum was determined by filtrating 15 mL of the activated sludge over a preweighed 12 μm cellulose nitrate filter. This filter was dried for 1.5 hour at 105.0 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 10.7 g/L. The pre-exposed sludge was homogenized before use by pressing it through a sterile needle with a syringe. The homogenized pre-exposed sludge was subsequently diluted further to 2 mg dry weight/L concentration in the bottles.
The Colony forming units (CFU) of the diluted homogenized unexposed and pre-exposed inocula was determined by a colony count method based on the ISO 6222 (1999) guideline. Both inocula as used in the closed bottles (2 mg/L dry weight) were diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 mL of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar. Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.7 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was for both the unexposed and pre-exposed activated sludge inocula 1.2E+06 CFU/L.

Duration of test (contact time):

60 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.



Nutrients and stocks
The test medium used in the Closed Bottle test contained per liter of water 8.50 mg KH2PO4,
21.75 mg K2HPO4, 33.41 mg Na2HPO4·2H2O, 22.51 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25
mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification
that is not related to the biodegradation of the test substance.
The test substance was added to the bottles from a stirred aqueous stock suspension of 1 g/L.
This stable homogenous white/opaline suspension of the test substance was prepared by
acidifying the test substance stock first to pH 3.0 using a 2 M HCl solution. The acidified
mixture was subsequently homogenized by stirring. Next, the pH of the homogenous
suspension was adjusted to pH 5.7 by using a 1 M NaOH solution and the final volume was
adjusted with demineralized water to obtain a 1 g/L test substance concentration. The stock
suspension was stirred at ambient temperature until use. The pH of the stock suspension was
5.6 when dosed to the test bottles. The reference substance sodium acetate was added to the
bottles using an aqueous stock solution of 1.0 g/L.

Test procedures:
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). For both inocula use was made of 10 bottles containing only the inoculum, 10 bottles containing the inoculum and silica gel, 10 bottles containing the inoculum and silica gel with test substance, 6 bottles containing the inoculum and sodium acetate. Silica gel (1 g) was added to the bottles and volumes of 0.6 mL of the test substance stock suspension were dosed directly to the bottles onto the silica gel. Next the bottles were filled with the nutrient solution and the inoculum and subsequently closed. The concentrations of the test substance and sodium acetate in the bottles were 2 mg/L and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of sodium acetate were withdrawn for analyses of the dissolved oxygen concentration at day 7 and 14. Two duplicate bottles of the control, the silica gel control and the test substance were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease at day 42, 60 and 84 using the bottles of day 28 and a special funnel.

Analyses
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). A special funnel was used that fitted exactly in the BOD bottle. To measure the oxygen concentration the oxygen electrode was inserted in the BOD bottles. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode, the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, 1992).
The pH was measured using an Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger.

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThODNH3

Value:

62

Sampling time:

84 d

Remarks on result:

other: test medium + 1 g silicagel

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThODNO3

Value:

58

Sampling time:

84 d

Remarks on result:

other: test medium + 1 g silicagel; *Biodegradation of two replicate bottles was 56% and 61%

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThODNO3

Value:

54

Sampling time:

60 d

Remarks on result:

other: test medium + 1g silica gel

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThODNO3

Value:

35

Sampling time:

42 d

Remarks on result:

other: test medium + 1g silica gel

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThODNO3

Value:

28

Sampling time:

28 d

Remarks on result:

other: test medium + 1g silica gel

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThODNH3

Value:

57

Sampling time:

60 d

Remarks on result:

other: test medium + 1g silica gel

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThODNH3

Value:

38

Sampling time:

42 d

Remarks on result:

other: test medium + 1g silica gel

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThODNH3

Value:

30

Sampling time:

28 d

Remarks on result:

other: Test medium + 1g silica gel

Details on results:

Theoretical oxygen demand (ThOD)
The ThODNH3 of the test substance used to calculate the biodegradation percentages was 2.88 g oxygen/g test substance and the ThODNO3 was 3.05 g oxygen/g test substance. This ThOD was calculated from the average molecular formula and molecular weight of the test substance. Biodegradation percentages were calculated using a 99.52% organic content for the test substance (0.48% was the water fraction – Annex 2).The ThOD of sodium acetate was 0.78 g oxygen/g sodium acetate.

Toxicity
Inhibition of the degradation of a well-degradable substance, e.g. sodium acetate by the test substance in the Closed Bottle test is optional in the OECD 301D guideline and was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition can be detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of the test substance (higher oxygen concentration in bottles with test substance compared to the concentration in the control bottles). Inhibition of the endogenous respiration by the test substance at day 7 was not detected for both inocula (Table I). Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Test conditions
At the start of the test with the unexposed activated sludge the pH of the media in the control bottles, the acetate reference bottles, the control bottles with silica gel and the test substance bottles were 7.0, 6.8, 6.9, and 6.9, respectively. The pH of the medium in the duplicate reference bottles with the unexposed activated sludge measured at day 14 was 6.7. The pH of the medium in the duplicate bottles of the tests with unexposed activated sludge at day 84 was 6.9 for the control bottles, pH 6.4 and 6.3 for the test substance bottles, and pH 6.7 and 6.6 for the control bottles with silica gel.
The pH of the medium in the duplicate bottles of the tests with pre-exposed activated sludge at the start of the test was 7.0 for the control bottles, pH 7.0 and 6.9 for the test substance bottles, pH 6.9 for the control bottles with silica gel and pH 7.0 for the acetate reference bottles.
The pH of the medium in the duplicate reference bottles with the pre-exposed activated sludge measured at day 14 was 6.8. The pH of the medium in the duplicate bottles of the tests with pre-exposed activated sludge at day 84 was 7.1, 6.5 and 6.6 for the control bottles, the test substance bottles, and the control bottles with silica gel, respectively.
The temperature ranged from 22.5 to 22.9 °C, which is within the prescribed temperature range of 20 to 24°C.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration that does not not exceed the 1.5 mg/L criterion for the endogenous respiration and was 1.00 mg/L and 0.70 mg/L at day 28 for the unexposed and pre-exposed activated sludge inoculum, respectively (Table I). The biodegradation percentage of the reference substance, sodium acetate, at day 14 was 88% and 68% for the unexposed and pre-exposed activated sludge inoculum, respectively. The biodegradation percentage of the reference substance herewith reached the pass level of 60% biodegradation at day 14 for both inocula (Table II, Figure 1 and Table III, Figure 2). Furthermore, the differences of extremes of the duplicate values at the end of both tests were less than 20%. Finally, the validity of both tests is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period (Table I).

Biodegradability
Assuming a complete nitrification of the test substance organic nitrogen (biodegradation based on the ThODNO3) the test substance was biodegraded by 28% and 27% in the Closed Bottle test at day 28 inoculated with unexposed and pre-exposed activated sludge, respectively (Table II, Figure 1 and Table III, Figure 2). The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 54% and 41% at day 60 using unexposed and pre-exposed activated sludge as inoculum, respectively (Table II, Figure 1 and table III, Figure 2). The biodegradation of <60% demonstrates that 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol did not ultimately (completely) biodegrade within 60 days. A biodegradation percentage <60% at day 60 does not allow a classification as not persistent.
The test with the pre-exposed inoculum was expected to show a faster biodegradation of the test substance due to the presence of more competent microorganisms. However, the biodegradation of both the test substance and the reference substance was for unknown reasons slower with the pre-exposed activated sludge compared to the unexposed activated sludge.
Mineralization (>60% biodegradation) of the 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol was demonstrated with the unexposed activated sludge inoculum at day 84 of the test and using the ThODNH3 to calculate the biodegradation (Table II). When the complete nitrification of the test substance organic nitrogen was assumed only one out of the two replicates achieved >60% biodegradation at day 84 (Table II). These results suggest that 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol is ultimately biodegradable and the lack of a “fast” ultimate biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

Results with reference substance:

The biodegradation percentages of the reference substance herewith reached the pass level of 60% biodegradation at day 14 for both inocula.

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Unexposed activated sludge				Pre-exposed activated sludge
	Mcs	Mts	Mc	Ma	Mcs	Mts	Mc	Ma
0	8.9	8.9	8.9	8.9	8.9	8.9	8.9	8.9
	8.9	8.9	8.9	8.9	8.9	8.9	8.9	8.9
Mean (M)	8.90	8.90	8.90	8.90	8.90	8.90	8.90	8.90
7	8.5	8.3	8.5	4.3	8.5	7.8	8.6	6.1
	8.5	8.3	8.5	4.3	8.5	7.7	8.6	6.1
Mean (M)	8.50	8.30	8.50	4.30	8.50	7.75	8.60	6.10
14	8.3	7.6	8.4	3.9	8.3	7.3	8.4	4.9
	8.3	7.5	8.4	3.7	8.4	7.3	8.5	4.9
Mean (M)	8.30	7.55	8.40	3.80	8.35	7.30	8.45	4.90
21	8.0	7.0	8.0		8.2	6.8	8.3
	8.0	7.0	8.1		8.1	6.6	8.3
Mean (M)	8.00	7.00	8.05		8.15	6.70	8.30
28	7.8	6.2	7.9		8.1	6.3	8.2
	7.9	6.1	7.9		7.9	6.4	8.2
Mean (M)	7.85	6.15	7.90		8.00	6.35	8.20
42	7.7	5.7	7.9		8.0	5.9	8.1
	7.7	5.4	7.9		7.8	5.8	8.0
Mean (M)	7.70	5.55	7.90		7.90	5.85	8.05
60	7.6	4.2	7.8		7.6	5.3	7.9
	7.6	4.5	7.8		7.9	5.2	7.8
Mean (M)	7.60	4.35	7.80		7.75	5.25	7.85
84	7.4	3.7	7.7		7.6	5.1	7.8
	7.4	4.0	7.6		7.9	5.2	7.7
Mean (M)	7.40	3.85	7.65		7.75	5.15	7.75

Mcs         Mineral nutrient solution with inoculum and silica gel

Mts          Mineral nutrient solution with inoculum, silica gel, and test substance (2.0 mg/L)

Mc           Mineral nutrient solution with only inoculum

M_a      Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L)

 

 

Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test inoculated with unexposed activated sludge. Biodegradation of the test substance is calculated both without nitrification correction (BOD/ThOD_NH3) and with nitrification correction (BOD/ThOD_NO3)

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance		Acetate
			ThODNH3	ThODNO3
0	0.00	0.00	0	0	0
7	0.20	4.20	3	3	80
14	0.75	4.60	13	12	88
21	1.00		17	16
28	1.70		30	28
42	2.15		38	35
60	3.25		57	54
84	3.55		62	58*

*Biodegradation of two replicate bottles was 56% and 61%

Table III Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test inoculated with pre-exposed activated sludge. Biodegradation of the test substance is calculated both without nitrification correction (BOD/ThOD_NH3) and with nitrification correction (BOD/ThOD_NO3)

Time (days)		Oxygen consumption (mg/L)		Biodegradation (%)
		Test substance	Acetate	Test substance		Acetate
				ThODNH3	ThODNO3
0		0.00	0.00	0	0	0
7		0.75	2.50	13	12	48
14		1.05	3.55	18	17	68
21		1.45		25	24
28		1.65		29	27
42		2.05		36	34
60		2.50		44	41
84		2.60		45	43

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Remarks:

(28% after 28 days, based on ThODNO3); but (54 % after 60 days- prolonged exposure): further degradation expected because a plateau has not been reached.

Conclusions:

Under the study conditions, the test substance was determined to be inherently biodegradable with 54% biodegradation (based on ThODNO3) after 60 days (and 58% biodegradation after 84 days)

Executive summary:

To assess the biotic degradation of 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol a ready biodegradability test was performed, which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines.

The biodegradability of the test substance (2.0 mg/L) was assessed using unexposed and pre-exposed activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles, and incubated in the dark at 22.5 to 22.9°C for 84 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, the test item did not cause a reduction in the endogenous respiration of the unexposed and pre-exposed activated sludge at day 7. The test substance is therefore considered to be non-inhibitory to both inocula.

Assuming a complete nitrification of the test substance organic nitrogen (biodegradation based on the ThOD_NO3) the test substance was biodegraded by 28% and 27% in the Closed Bottle test at day 28 inoculated with unexposed and pre-exposed activated sludge, respectively (Table II, Figure 1 and Table III, Figure 2). The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 54% and 41% at day 60 using unexposed and pre-exposed activated sludge as inoculum, respectively (Table II, Figure 1 and table III, Figure 2). The biodegradation of <60% demonstrates that 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol did not ultimately (completely) biodegrade within 60 days. A biodegradation percentage <60% at day 60 does not allow a classification as not persistent. Mineralization (>60% biodegradation) of the 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol was only demonstrated with the unexposed activated sludge inoculum at day 84 of the test. The lack of a “fast” ultimate biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

The test with the unexposed and pre-exposed activated sludge inoculum are both valid as shown by an endogenous respiration of 1.0 mg/L and 0.70 mg/L, respectively. By the total mineralization of the reference substance, sodium acetate. Sodium acetate was degraded by  88% using the unexposed activated sludge inoculum and by 68% using the pre-exposed activated sludge inoculum. Furthermore, the differences of extremes of the duplicate values at the end of the test were less than 20% for both inocula. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Description of key information

Di (2-hydroxypropyl) tallow amine (PFAPO T) is at normal test substance concentrations toxic to the microorganisms in the inoculum. Silica gel may be added to reduce the test substance concentration in the aqueous phase and facilitating slow release of the test substance. 

Several tests are available, which assessed the biodegradability and the removal potential of  Di (2-hydroxypropyl) tallow amine (PFAPO T):

 

Study I (van Ginkel, 2009, study number T090942 C): 

For the closed bottle test unadapted river water and 2 g/bottle silica gel were used to mitigate the toxicity of the test substance Di (2-hydroxypropyl) tallow amine (PFAPO T, Old CAS 68951-72-4).  The test substance concentration was 2 mg/L. In the closed bottle test (OECD TG 301D), the test substance was found to be readily biodegradable since 62% (based on ThODNH3) biodegradation was observed after 28 days. After correction of nitrification biodegradation percentage was 59% (based on ThODNO3). This value is slightly below the threshold value of 60% after 28 days, but based on the observations in study II, it can be considered that Di (2-hydroxypropyl) tallow amine is readily biodegradable. 

 

Study II (van Ginkel, 2009, document number ECRA 2.391.558): 

For the closed bottle test unadapted river water and 2 g/bottle silica gel were used to mitigate the toxicity of the test substance Di (2-hydroxypropyl) tallow amine (PFAPO T, Old CAS 68951-72-4).  The test substance concentration was 1 mg/L. In the closed bottle test (OECD TG 301D), the test substance was found to be readily biodegradable since 71% (based on ThODNH3) and 67% (based on ThODNO3) biodegradation was observed after 28 days. Furthermore, in the SCAS test activated sludge was exposed to a specified concentration of the test substance and the non-purgeable organic carbon (NPOC) was analysed frequently to determine the carbon removal. The calculated organic carbon concentration of PFAPO T in the influent of the SCAS test was 37.2 mg/L. The measured concentrations of the NPOC in the filtrated and unfiltrated dilutes stock solutions (1:20) was 33.5 and 34.0 mg/L. 34 mg/L (C_T) was used to calculate the removal percentages. At the end of the test the removal of di (2-hydroxypropyl) tallow amine increased to a level of >90% indicating degradation of the hydrophilic moiety. Di (2-hydroxypropyl) tallow amine (PFAPO T) is a surfactant consisting of a hydrophilic part i.e. di (2-hydroxypropyl) amine linked to a hydrophobic tallow-alkyl moiety. Biodegradation of both moieties of surfactants usually requires the concerted action of at least two species of microorganisms as one single micro-organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). The biodegradation mechanism of di (2-hydroxypropyl) tallow amine was deduced from semi-continuous activated sludge (SCAS) test results (test report ECRA 2.391.558; Ginkel et al. 2009).

In the SCAS test activated sludge was exposed to a specified concentration of the test substance (1 g/L) and the non-purgeable organic carbon (NPOC) was analysed frequently to determine the carbon removal. This test has been performed according to OECD Test Guidelines (OECD, 1981). The calculated organic carbon concentration of di (2-hydroxypropyl) tallow in the influent of the SCAS test was 37.2 mg/L. The measured concentrations of the non purgeable organic carbon (NPOC) in the filtrated and unfiltrated diluted stocks (1:20) was 33.5 mg/L and 34.0 mg/L, respectively. 34.0 mg/L (C_T) was used to calculate the removal percentages. The test substance caused no reduction of the removal of organic carbon present in the primary settled sewage. Di (2-hydroxypropyl) tallow amine present at a concentration of 50 mg/L is therefore considered to be non-inhibitory to the activated sludge present in full-scale biological treatment plants. These test conditions allow biodegradation of di (2-hydroxypropyl) tallow amine by microorganisms present in activated sludge.

Effluent NPOC values obtained from the test substance and control units were comparable and constant before additions of di (2-hydroxypropyl) tallow amine. During the first 10 days, the carbon removal ranged from 60 to 72% suggesting the formation of hydrophilic compound(s). Thereafter the NPOC values of effluent of the unit fed with domestic wastewater spiked with di (2-hydroxypropyl) tallow amine did decrease further. During the last weeks of the test the removal of di (2-hydroxypropyl) tallow amine increased to a level of >90% indicating degradation of the hydrophilic moiety.The most likely mechanism derived from the SCAS test results is the oxidation of the hydrophobic part (alkyl chain) from the far-end. This oxidation leads to formation of water-soluble amines with two 2-hydroxypropyl and an acetate or butyrate group. These water-soluble amines with two 2-hydroxypropyl and an acetate or butyrate group represent approximately 40% of the carbon of the surfactant. The partial degradation of the alkyl chain and the hydrophilic moiety are degraded by a consortium of microorganisms.

The high carbon removal percentages after an initial partial removal strongly indicate that di (2-hydroxypropyl) tallow amine is ultimately biodegradable. Closed Bottle tests inoculated with adapted activated sludge from the SCAS test are suitable to predict ultimate biodegradation of di (2-hydroxypropyl) tallow amine, which is not in the aqueous phase because of their capacity to adsorb. In the supporting study from van Ginkel et al. (study report ECRA 2.391.558, 2009) a Closed Bottle test with adapted sludge, di (2-hydroxypropyl) tallow amine is biodegraded 63% (based on ThODNH3)/60 % (based on ThODNO3) and also 71% (based on ThODNH3)/ 67% (based on ThODNO3) with unadapted sludge at day 28.  The biodegradation percentage in excess of 60% also indicates ultimate (complete) degradation. Also in the key study from van Ginkel 2009 (test report number 2.391.360) di (2-hydroxypropyl) tallow amine (PFAPO T) were 63% biodegraded (based on ThODNH3) in the presence of silica gel after 28 days.

 

The biodegradation is jeopardized by the toxicity of the test substance and with the addition of silica gel the release of the substance to the aqueous phase is slow. Therefore, the mineralization in a closed bottle test (OECD TG 301D) is slowed down and the biodegradation of more than 60% might need longer than 28 days. However, inhibition of the biodegradation in OECD tests is an artefact because environmental concentrations are in the range of µg/L and ultimate biodegradation was also shown in the SCAS test.  

 

In principle the two moieties of this fatty amine derivative are degraded sequentially. The degradation curve will therefore be the sum of two growth curves. The biodegradation of the two moieties may be fully in line with the time window criterion (10-day) when judged as separate chemicals. The time window criterion was developed on the assumption that a compound is degraded according to the “standard” growth curve in ready biodegradability tests. The time-window should therefore be ignored as a pass fail criterion for this surfactant in ready biodegradability tests.

 

References: 

Van Ginkel CG. Complete degradation of xenobiotic surfactants by consortia of aerobic microorganisms. Biodegradation. 1996 Apr;7(2):151-64.

Study III (Geerts, 2023, document number: 20221033; study number:T22024 C):

To assess the biotic degradation of 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol a ready biodegradability test was performed, which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines using unexposed and pre-exposed activated sludge as inoculum and 1 g/bottle silica gel to mitigate the toxicity of the test substance.

The biodegradability of the test substance (2.0 mg/L) was assessed using unexposed and pre-exposed activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles, and incubated in the dark at 22.5 to 22.9°C for 84 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, the test item did not cause a reduction in the endogenous respiration of the unexposed and pre-exposed activated sludge at day 7. The test substance is therefore considered to be non-inhibitory to both inocula.

Assuming a complete nitrification of the test substance organic nitrogen (biodegradation based on the ThOD_NO3) the test substance was biodegraded by 28% and 27% in the Closed Bottle test at day 28 inoculated with unexposed and pre-exposed activated sludge, respectively (Table II, Figure 1 and Table III, Figure 2). The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 54% and 41% at day 60 using unexposed and pre-exposed activated sludge as inoculum, respectively (Table II, Figure 1 and table III, Figure 2). The biodegradation of <60% demonstrates that 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol did not ultimately (completely) biodegrade within 60 days. A biodegradation percentage <60% at day 60 does not allow a classification as not persistent. Mineralization (>60% biodegradation) of the 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol was only demonstrated with the unexposed activated sludge inoculum at day 84 of the test. The lack of a “fast” ultimate biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

The test with the unexposed and pre-exposed activated sludge inoculum are both valid as shown by an endogenous respiration of 1.0 mg/L and 0.70 mg/L, respectively. By the total mineralization of the reference substance, sodium acetate. Sodium acetate was degraded by  88% using the unexposed activated sludge inoculum and by 68% using the pre-exposed activated sludge inoculum. Furthermore, the differences of extremes of the duplicate values at the end of the test were less than 20% for both inocula. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Conclusion: 

Although the % biodegradation (based on ThODNO3) was slightly below 60% assuming a complete nitrification of the organic nitrogen present in the test substance in study I it was initially considered justified to classify Di (2-hydroxypropyl) tallow amine (PFAPO T) as readily biodegradable also based on the results presented in study II. Study III however shows that based on the linearity of the biodegradation curve that the biodegradation rate is limited by the bioavailability of the substance. This biodegradation rate is however not high enough to classify the substance readily biodegradable. With a biodegradation percentage 54% on day 60 it is concluded to classify the substance as inherently biodegradable.

 

 

Further information can be found in the read-across justification in chapter 13. 

 

 

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Justfiication of the omission of ammonium chloride from the mineral salts medium of the OECD 301D ready biodegradation test

The ready biodegradation test medium
Ammonium chloride is added to the standard ready biodegradation test (RBT) medium as macro-nutrient for the growth of the microorganisms that are responsible for the biodegradation of the test substance. Ammonium chloride is added in RBT tests under the assumption that there is a nitrogen limitation in RBTs. The OECD 301D test uses however the lowest test substance concentration of all RBTs and the omission of ammonium chloride does not result in nitrogen limitation as shown by the biodegradation of the reference compound in these tests.
Ammonium chloride added with the medium in the OECD 301D test results in an excess of ammonium in the test which will be oxidized to nitrate. Nitrification is performed by a small group of autotrophic bacteria which are not involved in the mineralization of the test substance. The test substance will be mineralized by heterotrophic bacteria. Adding ammonium chloride to the standard ready biodegradation test medium, not needed for growth of the heterotrophic bacteria, results only in growth of the nitrifying autotrophic bacteria.

Stringency of RBTs
A positive RBT result informs about three aspects of biodegradation: i) the presence and pervasiveness of competent microorganisms in the environment; ii) the ultimate biodegradation of the test substance; and iii) rates of biodegradation in the environment. RBTs are regarded as the most stringent biodegradation tests because biodegradation needs to be achieved within a certain time frame using a relatively small inoculum and a relative high concentration of one test substance.
Meaning, in the relatively small RBT inoculum already enough competent microorganisms should be present to achieve the mineralization of the test substance in the required time frame. The inoculum concentration required for ready biodegradation test is described in the OECD guidelines and is measured at the start of an RBT. Hence, the stringency of RBTs is confirmed (checked) at the start of the RBT.
Organic substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing the substance as carbon and energy source. The ammonium present in the standard RBT medium is oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) and are not involved in the biodegradation of organic substances. Hence, the numbers of nitrifying bacteria in the inoculum of RBTs do not affect/influence the stringency of the tests.

Accuracy of RBTs
In RBTs the respiration of the inoculum blank (the endogenous respiration*) is used as the value for the background respiration. On top of this background respiration, it should be possible to accurately measure the respiration by the test substance. In the OECD 301D test there is only a maximum amount of oxygen available at the start of the OECD 301D test (~9 mg O2/L at 20°C, saturation of oxygen in water). Oxygen concentrations in the test should stay aerobic (≥ 0.5 mg O2/L) and a maximum endogenous respiration of 1.5 mg O2/L is allowed. This means that there is ~7 mg O2/L left for the biodegradation assessment of the test substance. If the endogenous respiration would use more oxygen there is less oxygen available to assess the biodegradation of the test substance resulting in a less accurate biodegradation assessment. The validity criteria of the inoculum blank therefore ensures the accuracy of the measured oxygen consumption by the test substance.

* Endogenous respiration is defined as: a situation in which living organisms oxidize some of their own cellular mass instead of oxidizing substrates they take from the environmental matrix.

The influence of the nitrification of the ammonium nitrogen supplemented with the mineral salts medium on the accuracy validity criteria was compared for the different respirometric RBTs. The biodegradation assessment in the OECD 301B test is based on the measurement of evolved CO2 and the accuracy of the test is therefore not affected by the nitrification of the ammonium nitrogen in the mineral salt medium. The OECD 301C, 301D and 301F are respirometric tests based on measurement of the oxygen consumption. Nitrification in the control bottles will in these tests be mistaken for endogenous respiration. Assuming a complete oxidation of the mineral salts ammonium nitrogen results in an additional oxygen consumption of 0.6 mg/L in the OECD 301D test and of 6 mg/L in the OECD 301C and 301F test. The contribution of this oxygen consumption is 40% of the allowed endogenous respiration in the OECD 301D test and is only 10% of the allowed endogenous respiration in the OECD 301C and 301F test. For the OECD 301D test it is known (see first paragraph above) that nitrogen is not limited in the test and therefore the supplemented ammonium nitrogen is an excess that will be oxidized to nitrate. In the OECD 301C and 301F a higher test substance concentration is used and therefore the nitrogen is expected to be limited to biodegrade all the test substance. In these tests part of the dosed mineral salts ammonium nitrogen will be incorporated in new biomass and the actual oxygen consumption by the nitrification is therefore expected to be < 6 mg/L. The contribution of the oxygen consumption of the allowed endogenous respiration in the OECD 301C and 301F will be <10%.

In conclusion: The back-ground respiration of the other RBTs is not or only slightly influenced by the addition of ammonium in the mineral salts medium compared to the influence it has on the back-ground respiration in the OECD 301D test.

High level of variation in RBT results.
The amount and rate of oxygen consumption by the nitrification of the ammonium chloride dosed with the mineral salts medium will mainly depend on the initial numbers of nitrifying bacteria present in the inoculum. These numbers will vary throughout the year because of the seasonal changes. Nitrifying bacteria are sensitive and relative slow growing bacteria. Low bacteria numbers at the start of the tests or an initial delay in growth by toxic effects is therefore not easily to overcome over a 28 days test period. A test substance that is (slightly) toxic to nitrifying bacteria will delay or stop the growth of nitrifying bacteria in the test bottles. In such a case the inoculum blank (with no hampering of the growth of nitrifying bacteria) will overestimate the background respiration in the test bottles resulting in lower (false) biodegradation values. Analysis of formed nitrate and nitrite in the OECD 301D test and control bottles allow a correction for the additional oxygen consumption. These analyses will however also introduce analytical inaccuracy and hence an increased variation (inaccuracy) of the final calculated test substance biodegradation.
The endogenous respiration in the OECD 301D test medium without addition of ammonium chloride and using bacterial densities (cells/L) in the prescribed range of the test guideline varies in general in the range of 1 ± 0.2 mg/L. The addition of ammonium nitrogen in the mineral salts medium would result in max 0.6 mg/L addition oxygen consumption and could therefore result in failing the endogenous respiration validity criteria. The result of adding ammonium chloride in the OECD 301D will therefore result in a higher chance of invalidating test results.

In summary:
An accurate and stringent assessment of the biodegradation potential of a test substance in the OECD 301D test is possible by omitting the ammonium chloride from the RBT test medium. The omission of ammonium chloride from the OECD 301D medium is justified because:
• There is no nitrogen limitation for the growth of heterotrophic micro-organisms in the OECD 301D test and therefore no additional nitrogen source needs to be added with the medium;
• The omission of ammonium chloride does not affect the stringency of the test because the initial bacterial density at the start of the test is demonstrated to be in the prescribed range;
• The omission of ammonium chloride improves the accuracy of the measured oxygen consumption by the test substance.
• It is much more difficult in the OECD 301D test compared to the other RBTs to fulfill the accuracy criteria when ammonium chloride is added (“biased” effect of ammonium between RBTs).
• Supplementing ammonium chloride in the mineral salts medium of the OECD 301D test will introduce a higher variability and more invalidated test results caused by nitrifying bacteria which are not involved in the biodegradation of the test substances.

Based on the above the omission of ammonium chloride from the OECD 301D test medium should be accepted. In addition, test results from OECD 301D tests where ammonium chloride was omitted from the medium are also accepted in other regulations (a.o. biocidal product directive, ecolabeling, etc…).

 

Justification for the use of river water as test medium:

According to OECD TG 301 it is clearly stated that an alternative source for the inoculum like surface water (e.g. river water) can be used for the test. Furthermore, also the
REACH guidance on Information Requirements and Chemical Safety Assessment (2017) mentions and justifies that micro-organisms (~10^5 cells/mL) in surface waters can be used as inoculum for the closed bottle test.
In principle, organic chemicals should be introduced in all vessles (bottles) of OECD TG 301 and OECD TG 310 irrespective of the inoculum used. The biodegradable organic carbon introduced should in all cases be limited in order to guarantee a low respiration by the microorganisms introduced. Endogenous respiration (oxidation of storage material and protein) by the micro-organisms introduced with inocula is the major controbutor and should be <= 1.5 mg/L at day 28 in the closed bottle test (validity criterion). The endogenous respiration in the blank control bottles (river water) was 1.1 mg/L in study I and 1.3 mg/L in study II at day 28. When this validity criterion is met, it is shown that the test substance is the major source of carbon for energy and growth in the test.

 

Justification for data on the inoculum concentration: 

OECD tests of the 301 series and OECD 310 are characterized by their low inoculum concentration which mainly determines their stringency. A correct biomass concentration in the OECD 301 series and OECD 310 test is guaranteed by the validity criterium limiting the (endogenous) respiration in the control bottles. The endogenous respiration in the control is therefore a measure of the biomass concentration.

In the OECD 301D test the prescribed biomass concentration is in the range of 10⁴ – 10⁶ cells/L. The biodegradation of di (2-hydroxyproply) tallow amine (new CAS 1305599-79-0, old CAS 68951-72-4) was tested using the OECD 301D test with particle free river Rhine water (sampled from the river Rhine near Heveadorp, the Netherlands) as inoculum in two different studies. The river water was aerated for 7 days before use to reduce the endogenous respiration. No information on the actual cell numbers in the inoculum are reported for both studies, however, the endogenous respiration in study I was 1.1 mg/L and 1.3 mg/L in study II at day 28 which suggests that the biomass concentration was in the correct range.

Table 1 below shows an overview of measured endogenous respirations and the corresponding inoculum concentrations, determined by colony count method (ISO 6222, 1999), in OECD 301D GLP tests that were performed with particle free river Rhine water. The river water in these studies was sampled from the same place, River Rhine near Heveadorp (the Netherlands), as the GLP study and the non-GLP study for di (2-hydroxyproply) tallow amine. All river water samples were also aerated for 7 days before use in order to reduce the endogenous respiration. The endogenous respiration from all these river water samples ranged from 0.75 – 1.50 mg/L and the inoculum concentration ranged from 6E 10⁴ to 1E 10⁶ CFU/L. These results demonstrate that the river water fulfilled the endogenous respiration validity criterium and contained a biomass concentration that is in the prescribed range of the OECD 301D TG. The measured endogenous respiration in the OECD 301D test with di (2-hydroxyproply) tallow fulfilled the validity criterium (<1.5 mg/L at day 28) and therefore it can be concluded that the tests also contained a biomass concentration in the prescribed range of the OECD 301D test guideline.

 

 

Table 1 Overview of measured endogenous respirations and the corresponding inoculum concentration in particle free river Rhine water sampled near Heveadorp (the Netherlands) from different OECD TG 301D studies throughout 2020 – 2022 under GLP conditions

OECD 301D GLP study	Date sampling river water	Endogenous respiration day 28 (mg/L)	Inoculum concentration (colony forming units/L)
T20007 C	02-Mar-2020	1.25	9E 10^5
T20009 C	30-Mar-2020	0.75	9E 10^5
T20015 C	11-May-2020	1.50	1E 10^6
T20018 C	29-Jun-2020	1.45	1E 10^6
T20019 C	14-Sep-2020	1.05	5E 10^5
T20023 C	12-Oct-2022	1.05	1E 10^6
T20026 C	09-Nov-2020	0.95	9E 10^5
T20027 C	09-Nov-2020	0.95	9E 10^5
T21001 C	01-Mar-2021	1.00	1E 10^6
T21004 C	05-Apr-2021	1.45	4E 10^5
T21015 C	21-May-2021	1.00	4E 10^5
T21019 C	28-Jun-2021	1.40	5E 10^5
T22006 C	21-Mar-2022	1.45	1E 10^6
T22016 C	27-Jun-2022	1.45	6E 10^4