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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-Nov-2022 until 09-Jan-2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2009
Deviations:
no
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
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…).




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 1.2 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: freshwater
Remarks:
River water was sampled from the Rhine near Heveadorp, The Netherlands (28-Nov-2022). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (28-Nov-2022).
The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km
upstream. 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 after 1 day while moderately aerating.
The Colony forming units (CFU) in the particle free and preconditioned river water was
determined by a colony count method based on the ISO 6222 (1999) guideline. The
preconditioned river water used in the closed bottles was 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 of 22.7 °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 4.4E+05 CFU/L.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
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 river water 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 emulsion of 1 g/L with a pH of 5.7. This homogenous and stable emulsion 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 emulsion was adjusted to pH 5.6 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 emulsion was stirred at ambient temperature until use. 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). Use was made of 10 bottles containing only river water incubated with a regular stirring of the content, 10 bottles containing river water and the test substance incubated with a regular stirring of the content, 6 bottles containing river water and sodium acetate and 10 bottles containing only river water. The content of the bottles was stirred 10 times per day for 5 minutes to improve the bioavailability of the test substance in the bottles. The regular stirring of the content in the bottles was achieved by using a stirrer bar and stirrer plate together with a time switch to start and stop the stirrer plate.
Volumes of 0.6 mL of the test substance stock emulsion were added directly to the bottles already filled with nutrient spiked river water and subsequently closed. The concentrations of the test substance and sodium acetate in the bottles were 2.0 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 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.
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
based on ThODNO3
Value:
66
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Remarks:
based on ThODNH3
Value:
69
Sampling time:
28 d
Theoretical oxygen demand (ThOD) 

The ThODNH3 of the test substance used to calculate the biodegradation percentages was 3.31 g 

oxygen/g test substance and the ThODNO3 was 3.44 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 99.97% organic content for the test 

substance (0.03% 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 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 of the inoculum by the test substance at day 7 

was not detected (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 the pH of the media in the duplicate reference substance and control 

bottles (stirred and unstirred) was 8.1 and was 8.0 in the duplicate bottles containing the test 

substance. The pH of the medium in the duplicate reference bottles measured at day 14 was 

7.7. The pH of the medium in the duplicate control bottles (stirred and unstirred) at day 28 was 

8.0 and was 7.5 and 7.4 in the duplicate test substance bottles. The temperature ranged from 

22.6 to 22.7 °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 of 1.3 mg/L at day 28 in 

the control bottles which were stirred regularly (Table I) and does not exceed the 1.5 mg/L 

criterion for the endogenous respiration. The endogenous respiration at day 28 in the control 

bottles for which the content was not stirred was 1.2 mg/L. The endogenous respiration in the 

control bottles was therefore not, or only slightly, increased by the regular stirring of the 

content. The biodegradation percentage of the reference substance, sodium acetate, at day 14 

was 84% and herewith reached the pass level of 60% biodegradation at day 14 (Table II and 

Figure 1). Furthermore, the differences of extremes of the duplicate values at day 28 were less 

than 20%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all 

bottles during the test period (Table I). 

 

 

 

Biodegradability 

Amines, di-C16-C18-alkyl methyl was biodegraded by 69% (based on ThODNH3) at day 28 in 

the Closed Bottle test (Table II). Assuming complete nitrification as a worst-case and 

calculating the biodegradation with the ThODNO3 to correct for the oxygen consumption by the 

nitrification resulted in 66% biodegradation at day 28 (Table II, Figure 1). Over 60% 

biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle 

test) immediately following the attainment of 10% biodegradation. The time window criterion 

should however not be applied because amines, di-C16-C18-alkyl methyl is a mixture of 

different constituents. The biodegradation of the test substance is therefore an addition of 

different biodegradation curves. It is thus possible that individual compounds meet the time 

window criterion whereas the biodegradability curve of the mixture of constituents suggests 

that the test substance is not readily biodegradable. The time window is therefore an 

inappropriate pass/fail criterion for mixtures of chemicals (Richterich and Steber, 2001; EU 

CLP commission regulation No 286). Amines, di-C16-C18-alkyl methyl is classified as readily 

biodegradable based on the ≥60% biodegradation reached at day 28. 

 


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


























































































































Time (days)



Oxygen concentration (mg/L)



 



Mcs



Mts



Mc



Ma



0



9.0



9.0



9.0



9.0



 



9.0



9.0



9.0



9.0



Mean (M)



9.00



9.00



9.00



9.00



7


8.15.88.54.5

 


8.15.68.44.5

Mean (M)


8.105.78.454.50

14


8.14.38.23.8

 


8.03.98.33.9

Mean (M)


8.054.108.253.85

21


7.72.57.9-

 


7.82.98.0-

Mean (M)


7.752.707.95-

28


7.73.17.8-

 


7.73.27.8-

Mean (M)


7.703.157.80-

Mcs  River water with nutrients, bottle content regularly stirred


Mts  River water with nutrients and test substance (2.0 mg/L), bottle content regularly stirred


Mc  River water with nutrients


Ma  River water with nutrients and sodium acetate (6.7 mg/L)


 


 


Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of the sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThODNH3) and with nitrification (BOD/ThODNO3).





























































Time (days)



Oxygen consumption (mg/L)



Biodegradation (%)



Test substance



Acetate



Test substance



Acetate



ThODNH3



ThODNO3



0



0.00



0.00



0



0



0



7


2.403.95363576

14


3.954.40605784

21


5.05-7673-

28


4.55-6966-


 
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Remarks:
Assuming complete nitrification as a worst-case of the test substance organic nitrogen, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 66% at day 28 in the Closed Bottle test. Based on ThODNH3 the test substance was biodegraded by 69%.
Conclusions:
Under the study conditions, the test substance was determined to be classified as readily biodegradable based on ≥60% biodegradation reached at day 28 (66% biodegradation based on ThODNO3).
Executive summary:

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 test substance (2.0 mg/L) was exposed to river water, which was spiked to a mineral
nutrient solution, dosed in closed bottles, incubated in the dark at 22.6 to 22.7°C and the
content in the bottles was regularly stirred (10 times per day for 5-minute periods) for 28 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 at day 7. The test substance is therefore considered to be non-inhibitory to the
inoculum.
Assuming complete nitrification of the test substance organic nitrogen, and calculating the
biodegradation based on the ThODNO3 the test substance was biodegraded by 66% at day 28 in
the Closed Bottle test. Over 60% biodegradation was not achieved within a period of 10 days
(14 days for the Closed Bottle test) immediately following the attainment of 10%
biodegradation. The time window criterion should however not be applied because amines, di-
C16-C18-alkyl methyl is a mixture of constituents (UVCB). The biodegradation of the test
substance is therefore an addition of different biodegradation curves, and the time window
should not be used as a pass/fail criterion. Amines, di-C16-C18-alkyl methyl is classified as
readily biodegradable based on the ≥60% biodegradation reached at day 28.
The test is valid as shown by an endogenous respiration of 1.30 mg/L determined in the control
bottles that were incubated with a regular stirring of the content and by the total mineralization
of the reference compound, sodium acetate. Sodium acetate was degraded by 84% of its
theoretical oxygen demand after 14 days. Furthermore, the differences of extremes of the
duplicate values at day 28 were less than 20%. 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

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Ready biodegradability of Amines, bis(hydrogenated tallow alkyl) methyl has been investigated through OECD Guideline 301 D, modified according to the recommendations of ECETOC (1985) or Blok et al. using emulsifier.

Activated sludge were cultured for 28 days in presence of Amines, bis(hydrogenated tallow alkyl) methyl.

More than 86% biodegradation of test substance occured after 28 days. The substance was considered to be readily biodegradable.