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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Description of key information

Several types of information is available which in a WoE can cover the OECD TG 309 and OECD TG 308.

Activated sludge simulation test: equivalent to OECD TG 303A: In effluent the substance transforms into polar metabolites for 96% (log Kow < 2.1) including CO2 formation and uptake in biomass;

Simulation test in river water, equivalent to OECD TG 314: DT50 is < 1 day at 20°C and < 1.9 days at 12°C;

Simulation test in river sediment, equivalent OECD TG 308: DT50 is 9.5 days at 22°C and 17.4 days at 12°C;

Simulation test in soil, equivalent OECD TG 307: DT50 is 6 days at 22°C and 11 days at 12C.

Key value for chemical safety assessment

Half-life in freshwater:
1.9 d
at the temperature of:
12 °C
Half-life in freshwater sediment:
17.4 d
at the temperature of:
12 °C

Additional information

A half-life in water can be derived equivalent to an OECD TG 309 using a WoE based on several simulation studies, which are summarised below.

Activated sludge simulation test: In a continuous activated sludge study similar to OECD TG 303A (Schaefer and Cartee, Wildlife, sponsored by RIFM, 2009). [14C]-OTNE was applied to the test system to determine the removal rate, the biodegradation of OTNE and to determine the log Kow of its biodegradation products. Method: The test apparatus consisted of a single 3-liter aeration basin connected to a clarifier. The total volume of the system including the aeration basin, the clarifier, air-lift and connecting tubing was approximately 6 litre. [14C]-OTNE was administered to the aeration basin in combination with domestic sewage at a concentration of ca. 6 ug/l. The target hydraulic and solid retention were 6 hours and 10-day respectively. Samples were removed from the test vessels over a 39-day test period. The first days were used to assess the steady state and after the steady state the final fate of [14-C] was determined. Disappearance of parent and the formation of metabolites and their log Kows were determined by HPLC equipped with a radiochemical detector. The solids were combusted to determine the amount of radioactivity remaining with the solids. Results: The distribution of radioactivity in solvent extracts of the influent, effluent and mixed liquor was determined using HPLC. The resulting radio-chromatograms were divided into five regions of interest (ROI) as presented in Table 3. Table 3 also includes the percentage of the type of metabolite versus the % radioactivity found in the total effluent.

Region of Interest

Retention time (min)

Log Kow range based on references

Total effluent % of dosed radioactivity is

72.73 +/- 6.24%

Metabolite 1: early peaks



0.63 +/- 1.03%%

Metabolite 2: between peaks 1


< 2.1

7.00 +/- 0.99%

Metabolite 3: Degradation product 1


typical 6.05

< 2.1

62.01 +/- 6.5%

Metabolite 4: Between peaks 2


> 2.1< 6.5

0.7 +/-0.48%

Parent (OTNE)



2.39 +/- 0.54%

 Log Kow calculations revealed that [14C]-OTNE had a measured Log Kow of 6.63-6.86. Metabolite 4 has a log Kow range >2.1 but < 6.5. Excluding metabolite 4, 69.64% of the effluent radioactivity resulted in peaks with log Kow <2.1 and thus < 4.5.

 The average distribution of radioactivity during stead state (after 19 days) is presented in Table below: 72.73% in effluent, 4.45% wasted sludge, 8.39% CO2 and biomass and 6.3% volatilised, this results in a recovery of the total radioactivity of 92%. Based on this the % polar metabolites can be calculated.

 Total OTNE removal both from effluent and wasted solids is 89.72%. Of this % there is 69.87% (70.57-0.7% metabolite4) converted to polar metabolites (log Kow <2.1). The complete biodegradation (CO2 + biomass) is fully converted metabolism and can be added to this 69.87% resulting in 78.26 (polar metabolites + 8.39% CO2 and biomass). This means that total OTNE metabolism result in 87% polar metabolites. 

Total radioactivity

92.72 %(89.72 total removed +2.39 remaining parent)

Removal Mechanism

% Removed (avg of 15 samples)

Total Parent Removal

89.72+/-11.97 (remaining parent is 2.39%)

Conversion to Metabolites

70.57+/-6.26 (without % complete degradation)

Parent Removed by Sorption


Metabolites Removed by Sorption


Total Radioactivity Removal

30.08+/- 7.24


6.04+/- 0.99

Complete Biodegradation (CO2+ biomass)




 Distribution of [14C)-OTNE in the effluent during stead state are presented in the table below. Total removal of OTNE in effluent is 69.64% (72.73-2.39 parent -0.7% non-polar metabolite but excluding the 0.08% non-extractable). This means that of the total RA in effluent 96% are polar metabolites (72.73/ 69.64 (0.63+7+62.01).


% of Dosed Radioactivity

in Influent1


Total in extract

Parent (10.90-13.50 min)

Metabolite 1-Early Peaks (2.00-4.25 min)

Metabolite 2-Between Peaks 1 (4.26-6.51 min) Metabolite 3 Degradation Product 1 (6.52-8.10 min) Metabolite 4-Between Peaks 2 (8.11-10.89 min)



72.73 ± 6.24

 2.39 ± 0.54

0.63 ± 1.03

7.00 ± 0.99

62.01 ± 6.50

0.70 ± 0.48

0.08 ± 0.04 (not included in total extract)


Total in extract

Parent (10.90-13.50 min)

Metabolite 1-Early Peaks (2.00-4.25 min)

Metabolite 2-Between Peaks 1 (4.26-6.51 min)

Metabolite 3 Degradation Product 1 (6.52-8.10 min) Metabolite 4-Between Peaks 2 (8.11-10.89 min)


4.22 ± 1.24

3.99 ± 1.19


0.22 ± 0.07

0.01 ± 0.03

Non-extractable 2.35 ± 0.89; 1Values are the mean and standard deviation of 15 sampling dates during the steady state period.

Conclusion: In a continuous activated sludge study similar to OECD TG 303A with [14C]-OTNE 89% of the parent compound was removed. The radioactivity was attributed to metabolite conversion, mineralisation, volatile loss and loss due to sorption to solids.When looking solely in the effluent it can be seen that 96% of the total [14C]-OTNE found were more polar transformation products of OTNE. The transformation products, metabolite 1, 2 and 3(62.01+7+0.63%) showed Log Kow 2.1.

A die-away study in river water was carried out with radio-labelled C14 -OTNE (equivalent to OECD TG 314, Schaefer, Wildlife, sponsored by RIFM, 2006). Method: The test was carried out at a nominal concentration of 3 µg/l in a sample of river water with activated sludge added at a level of 4 mg SS/l (to mimic conditions in the mixing zone). The disappearance of parent material and the formation of metabolites were determined with radio thin layer chromatography (Rad-TLC). The temperature was maintained at 20±3ºC. Mass balances were made for the components in the test medium. Results: The parent substance disappears fast and four separate metabolites are formed in time at different rates and with higher polarity than the parent. The total radioactivity of metabolites rose from 25% after 5 minutes up to 89% on day 14. Circa 4% volatilised. After 28 days 10 % mineralisation to CO2 was detected.The DT50 (Disappearance 50) for primary degradation is <1 days at 20±3°C. Conversion to 12oC results in a DT 50 of < 1.9 days. The kinetic assessment showed an initial first-order loss rate of 1.83/h and a second first-order loss rate of 0.022/h.

Sediment simulation study equivalent to OECD TG 308: The fate of 14C-OTNE in sediment was studied in microcosms according to protocols described in documents from the U.S. Food and Drug Administration of 1987 (Envirogen, sponsored by IFF, 1999). Method: Samples were taken from the sediment of the Delaware River in central New Jersey, USA. Sealed flasks with sediment spiked with 17 µg test substance/g sediment dw (17 mg/kg) were incubated at laboratory ambient temperature for 12 weeks. Periodically the headspace was flushed for oxygen replenishment and the effluent gas was drawn through a train of scintillation fluids to capture volatile organics and CO2 to be determined by liquid scintillation counting. Periodically also flasks were sacrificed and exhaustively extracted with hexane/acetone. An aliquot of the solvent fraction was used for thin layer chromatography for analysis of the test substance and metabolites. The total 14C-mass balance was established for the parent and metabolites based on extraction as well as on combustion of the total matrix. Results: After 8 weeks circa 50% was recovered as 14-CO2 and < 1% remained of the parent material. The TLC results showed that the test substance was degraded to a range of more polar fractions. The total recovery of radiolabel from the solvent extraction was below 80% for the river sediment but with total combustion 74 - 84% was recovered showing that a significant portion of the radioactivity was strongly bound to the sediment matrix. After a lag time of approx. 7 days the initial rate of CO2 production was 0.8% /day. The half-life of the parent substance was estimated to be 9.5 days at 22°C.When converted to 12°C this DT50 results in 17.4 days.


ECHA requested in 2018 in a draft decision the OECD TG 309. The response to this draft decision is presented here to combine the available information and support the overall WoE.


Response LR to ECHA DD requesting a new OECD TG 309

A new OECD TG 309 test with radiolabelled OTNE will not elucidate any new information towards an overall PBT assessment of OTNE and its transformation products. Transformation of OTNE into polar metabolites occurs with log Kow < 2.1 and half-lives of the parent substance show < 1 day in water (23°C), 9.5 day in sediment (22°C) and 6 day in soil at ca 22oC. After conversion to 12oC these DT50s become 1.9, 17.4 and 11 days and these remain below the respective cut off criteria for P assessment. In addition log Kow information is available on the metabolites formed.

In the OECD TG 303A (Schaefer and Cartee, Wildlife, sponsored by RIFM, 2009) test the log Kow of the degradation products is determined showing that in effluent 96% of OTNE is transformed to CO2 and biomass (8.39%) and other into polar metabolites with Log Kow < 2.1. The other 4% is remaining OTNE and a non-polar metabolite: 2.37 and 0.7%, respectively.  


In the river Die Away test according to OECD TG 314 (Schaefer, Wildlife, sponsored by RIFM, 2006) a half-life of 1 day was seen at 20°C.The DT50 of the OTNE in surface water would result in < 1.9 days at 12°C. In sediment and soil compartment this will result in 17.4 and 11 days, respectively.

The recommendation in the ECHA DD is that an aerobic surface water-simulation biodegradation test (EU C.25./OECD 309) be performed, with the identification of transformation/degradation products. In the detailed explanation that justifies this need, the ECHA DD highlights several key points, outlined below:  

1.       The study performed in the natural sediment, entitled, “Fate of OTAN in Natural Environments” (Envirogen, 1999), is insufficient because it is not possible to draw a conclusion about the persistency of the registered substance; there is no information available in the study concerning the nature of the 14C not recovered (if it is about the parent substance and/or transformation/degradation products bound in the matrix);  

2.       The river water die-away study performed in 2006 by Schaefer (equivalent to an OECD TG 314-Simulation Tests to Assess the Biodegradability of Chemicals Discharged in Wastewater) is a “supporting study only”;  

3.       The study performed in the natural soils (both sludge amended and non-sludge amended), entitled, “Fate of OTAN in Natural Environments” (Envirogen, 1999) do not allow conclusions on persistency because of uncertainties about the behaviour of the substance in other soil types; and;  

4.       In all studies, the degradation/transformation products were not identified nor characterized.  

In response to these questions:

The following information puts the river water die-away study (OECD 314 equivalent) into a weight-of-evidence approach for non-P classification of OTNE and provides clarifications on the submitted persistency studies in reference to DD comments. In addition, this section highlights further evidence to justify low environmental concern of metabolites formed given their water-soluble nature in comparison to the parent compound.  

Clarifying mineralisation % in the provided persistency studies:  

The ECHA DD states that the performed study in natural sediment, entitled, “Fate of OTAN in Natural Environments” (Envirogen, sponsored by IFF, 1999), is insufficient for use because it is not possible to draw a conclusion about the persistency of the registered substance. According to ECHA there is no information available in the study concerning the nature of the 14C not recovered (if it is about the parent substance and/or transformation/degradation products bound in the matrix).  

LR-Response: In considering these comments, close examination of the study elucidated the cause of the non-recoverable fraction. Adjusting for the recoverable fraction demonstrates high levels of mineralization. The resulting yields, when corrected for the fraction recovered, are 79, 75 and 76% of the total recoverable radioactivity evolved as CO2 for sludge amended, and non-sludge amended soil and river sediment, respectively. This illustrates that in all cases, rapid and complete mineralization is occurring. The results are reliable when considering the losses due to non-extractable residues. This is based on the reasoning below.  

In the aerobic river sediment sample correcting for the non-recoverable fraction of 14C-OTNE activity resulted in 76% of the bioavailable OTNE associated with CO2 evolution, demonstrating complete mineralization. The low recovery of 14C in river sediment substrate was due to non-extractable residue formation. This was indicated by the sediment combustion study and inability of conventional solvents to extract the substance bound to the sediment matrix. On week 4, 6 and 8 of the OTNE fate study, 57, 44, and 39% of the radioactivity was associated with the sediment, as determined by sediment combustion. Over this period of time, as radioactivity associated with sediment decreased, the percent mineralized increased from 19 (bioavailable OTNE 76%-57%) to 37% (bioavailable OTNE 76%-39%). This is indicative of rate limited mineralization; as a portion becomes bioavailable it is subsequently mineralized. The lack of bioavailability can be attributed to a tightly bound sediment fraction. This was also seen in the soil samples. Complete mineralization was observed in aerobic soil transformation studies for OTNE, using sludge amended and non-sludge amended soils with CO2 evolution of 67 and 64%, respectively. Here, there was a greater recoverable fraction. The lack of a clear plateau associated with all matrices further indicates mineralization of remaining humic/soil bound fraction that would have continued beyond 12 weeks (84 days).  

In another study, the river water die-away study (equivalent to OECD TG 314, Schaefer, Wildlife, sponsored by RIFM, 2006), the rate and extent of biodegradation of 14C-OTNE in river water was also indicated. The purpose of this study was to understand the degradation kinetics in the wastewater mixing zone effluent and surface water. This is the point of entry for a “down the drain chemical” to the environment and therefore it is highly relevant as a line of evidence in the persistency assessment. High removal rates of the parent compound would suggest that any parent compound found in wastewater effluent would be rapidly transformed in stream.  In the river water-die-away study, the T1/2 for primary biodegradation of OTNE was determined to be < 1 day with more water-soluble intermediates being formed.

Considering transformation products in this river die-away OECD TG 314 study: The disappearance of the parent compound and the formation of metabolites were determined by radio thin layer chromatography (Rad-TLC). In table 5 of this river water die-away study, the Rf values of the Rad-TLC plate assay for the metabolites and the parent compound were reported. In this method, Rf is the ratio of the distance moved by the substance to the distance moved by the solvent on a chromatogram. Non-polar compounds move further up the plate resulting in an Rf value closer to 1, whereas polar substances spot earlier in the chromatogram and have an Rf value closer to 0. The metabolites in this study had Rf values that ranged from <0.000-.330 whereas the parent compound had an Rf= .543-.612, indicating that the products formed had greater water solubility.   Additional in silico information on transformation products considered for increasing the Weight of Evidence (will be included in the dossier update):  When the parent compound is modelled using the University of Minnesota’s Pathway Prediction System, it too shows metabolic products that have greater water solubility than the parent compound:  

Table OTNE, its modelled degradation products and their calculated log Kows and Water solubility



Metabolite 2

Metabolite 3

Metabolite 8






Log Kow (EpiSuite)





WS (EpiSuite)

1-10 mg/L




Here, the predicted metabolites from modelling show oxidation to alcohols as a primary metabolic transformation pathway. The lack of chemical specificity towards the oxidation position in OTNE renders Log Kow values nearly two log units lower than the parent compound, consistent with the persistency studies. This suggests that any metabolic oxidation product should have greater water solubility that should allow for additional metabolite generation leading to the disappearance of OTNE. Persistency study results in context of measured environmental concentrations: Using a Weight-of-Evidence approach, these studies demonstrate that OTNE is rapidly transformed into more polar, water soluble intermediates and is non-persistent. The continuous activated sludge study (Schaefer and Cartee, 2009) simulates wastewater treatment removal and demonstrates that the environment is likely to see water-soluble intermediates of OTNE and not the bulk of the parent compound (only 2.5% of total radioactivity in effluent found as parent compound). In agreement with Schaefer and Cartee (Wildlife, sponsored by RIFM, 2009), Bester et al. (2008) also observed high removal rates for OTNE in full scale treatment plants. It is likely that any remaining parent compound released in the effluent will be rapidly transformed. This is highlighted by the river water die-away study (Schaefer, Wildlife, sponsored by RIFM, 2006) and confirmed by studies that only measured ng-L concentrations of OTNE downstream of waste water outfalls (Klaschka et al., 2013). Even though OTNE is detected in surface waters, this is not unexpected given the volume of use. Even for highly removed compounds, high volume of use will result in measurable concentrations. Only under extreme rain events, where the treatment capacity of the local wastewater sewage authority is exceeded, would abnormally high concentrations of OTNE be measured (Corada-Fernandez et al., 2017). However, the instream concentrations due to these events would be considered short-lived and acute exposures. Comparing these concentrations to chronic PNECs would therefore be inappropriate.