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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: sediment simulation testing
Data waiving:
other justification
Justification for data waiving:
other:
Transformation products:
not measured
Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-20 - 2021-08-16 /// DRAFT
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Due to the tendency of the test substance to be lost by evaporation in typically used flow-through test systems, the guideline had to be adapted and a closed system has been used.
Justification for type of information:
This information is submitted based on ECHA communication/decision number SEV-D-2114341466-49-01/F.
Qualifier:
according to guideline
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The radiolabelled dimethyldiphenylether isomers were provided as separate solutions in methanol. The ratio of the single isomers in the application solution was adjusted according to the composition of the technical product. The initial application solution had a activity of 5905 kBq/mL.
Radiolabelling:
yes
Remarks:
14C-labelling of the phenyl ring(s)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
The study was conducted using lake water sampled by the testing facility. The surface water was sampled from Biggesee (Bigge drinking water reservoir), Germany (51°4’42” N, 7°50’3” E).

Sampling and storing details.
Name Biggesee lake water and sediment
Sampling location Biggesee lake, 57462 Olpe-Sondern, North-Rhine-Westphalia, Germany
Site description A freshwater lake fed by a stream from a weir on the river. Wood- and grassland around the lake.
Geographical region/ global co-ordinates Central Europe / 7°50’ East and 51°4’ Nord
Date of collection January 30, 2018, 10:00 to 12:00 a.m.
Sampling depth water 25 cm
Sampling depth sediement 50 cm below water surface
Collection procedure water Immersion of container
Collection procedure sediment Collection with a spade from upper layer of 10 cm
Duration of transportation (to test facility) 06 May 2019, 10:30 to 11:30 a.m.
Duration of storage (prior to use) 19 days at ≤8 °C

The sediment was sieved through a 2 µm mesh at the collection site. After receipt, the test water was filtered through a 100 µm mesh filter and stored above a sediment layer at ≤8 °C.

Physico-chemical properties of test surface water and sediment.
Surface water: Biggesee
Temperature at collection (oC) 5.7 °C
pH at collection 7.48
Oxygen concentration at collection (mg/L) 11.4
Oxygen concentration after filtration at date of application (mg/L) 7.6
pH after filtration at date of application 7.1
TOC water (mg/L) 7.02
Corg sediment (%) 2.89
DOC (mg/L) 7.52*
BOD (mg O2/L) 14.4
Nitrogen (total, mg/L) 6.2
Nitrate (mg/L) 9.0
Nitrite (mg/L) 0.029
Ammonium-N (mg/L) < 0.01
total P (λ=177.434 nm) (µg/L) 12.6
Dissolved Orthophosphate (total, mg/L) 0.12


Duration of test (contact time):
92 d
Initial conc.:
10 µg/L
Based on:
test mat.
Initial conc.:
100 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
Dimethyldiphenylether isomers were incubated in test water amended with small amounts of sediment using the test concentrations 10 µg/L and 100 µg/L for the isomer mixture under dark, aerobic conditions. The amount of the single isomers in the mixture was corresponding to the composition of the technical product. Sterile samples were additionally prepared to enable differentiation between biotic and abiotic degradation.
The mineralization of dimethyldiphenylether isomers was studied over an incubation time of 92 days at 12°C ± 2°C. Since the test substances are volatile, the test was conducted in a closed test setup.
Reference substance:
benzoic acid, sodium salt
Remarks:
14C radiolabelled
Test performance:
The test was performed according to the OECD-Guideline 309 "Mineralisation in Surface Water – Simulation Biodegradation Test" using a radiolabelled mixture of dimethyldiphenylether isomers. The mixture was prepared from the single isomers according to the composition of the technical product. The amounts of the single isomers in the mixture are summarized as follows:
Isomer CAS number % w/w in the mixture
2,2´-Dimethyldiphenylether 4731-34-4 5
2,3´-Dimethyldiphenylether 66658-61-5 28
2,4´-Dimethyldiphenylether 3402-72-0 12
3,3´-Dimethyldiphenylether 19814-71-2 27
3,4´-Dimethyldiphenylether 51801-69-5 23
4,4´-Dimethyldiphenylether 1579-40-4 5

Compartment:
natural water: freshwater
DT50:
>= 19 - <= 29.1 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: based on HPLC analysis
Remarks:
geometric mean of all isomers
Key result
Compartment:
natural water: freshwater
DT50:
>= 19.2 - <= 25.2 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: based on GC-MS analysis
Remarks:
geometric mean of all isomers
Compartment:
natural water: freshwater
DT50:
55.1 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,2'-Dimethyl-diphenylether
Remarks:
10 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
72.1 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,2'-Dimethylphenyl ether
Remarks:
100 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
19.2 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,3-Dimethyldiphenylether
Remarks:
10 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
33.1 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,3-Dimethylphenyl ether
Remarks:
100 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
20.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,4-Dimethylphenyl ether
Remarks:
10 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
28 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,4-Dimethylphenyl ether
Remarks:
100 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
16.8 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 3,3'-Dimethylphenylether
Remarks:
10 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
27.4 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 3,3'-Dimethylphenylether
Remarks:
100 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
16.8 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 3,4-Dimethyldiphenylether
Remarks:
10 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
21.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 3,4'-Dimethyldiphenylether
Remarks:
100 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
26.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 4,4'-Dimethyldiphenylether
Remarks:
10 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
19.4 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 4,4'-Dimethyldiphenylether
Remarks:
100 µg/L Level, determined by GC-MS
Compartment:
natural water: freshwater
DT50:
45.1 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,2'-Dimethyldiphenylether
Remarks:
10 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
65 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,2'-Dimethyldiphenyl ether
Remarks:
100 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
20.5 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,2-Dimethyldiphenylether
Remarks:
100 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
20.5 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,3'-Dimethyldiphenylether
Remarks:
10 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
32.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,3'-Dimethyldiphenylether
Remarks:
100 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
28.4 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,4'-Dimethyldiphenyl ether
Remarks:
10 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
29.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 2,4'-Dimethyldiphenyl ether
Remarks:
100 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
14.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 3,3'-Dimethyldiphenyl ether
Remarks:
10 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
26.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 3,3'-Dimethyldiphenylether
Remarks:
100 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
15.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 3,4'-Dimethyldiphenyl ether
Remarks:
10 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
22.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 3,4'-Dimethyldiphenylether
Remarks:
10 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
17.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 4,4'-Dimethyldiphenylether
Remarks:
10 µg/L Level, determined by HPLC-MS
Compartment:
natural water: freshwater
DT50:
21.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: 4,4'-Dimethyldiphenylether
Remarks:
100 µg/L Level, determined by HPLC-MS
Other kinetic parameters:
first order rate constant
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
Details on transformation products:
Formation of metabolites showed the degradation of dimethyldiphenylether isomers in surface water. During 92 days of incubation, the parent substance was degraded to several transformation products which were characterised by their retention time during HPLC analysis. In the organic extracts 8 unknown transformation product were detected. All of them were eluting earlier, indicating a higher polarity compared to the dimethyldiphenylether isomers. Three of the transformation products, signals detected at retention times of 17.6 min, 24.4 min and 25.8 min, exceeded 10 % AR in at least one of the concentration levels.
The maximum levels of the metabolite eluting after 17.6 min were 31 % AR after 92 d (concentration level of 10 µg/L) and 28.5 % AR after 76 d (concentration level of 100 µg/L). The maximum levels of the metabolite eluting after 24.4 min were 18.1 % AR after 42 d (concentration level of 10 µg/L) and 16.7 % AR after 626 d (concentration level of 100 µg/L). The maximum levels of the metabolite eluting after 25.8 min were 26.1 AR (concentration level of 10 µg/L) and 20.0 % AR (concentration level of 100 µg/L), in both cases observed after 42 d.
The maximum levels of the metabolite eluting after 17.6 min were 31 % AR after 92 d (concentration level of 10 µg/L) and 28.5 % AR after 76 d (concentration level of 100 µg/L). The maximum levels of the metabolite eluting after 24.4 min were 18.1 % AR after 42 d (concentration level of 10 µg/L) and 16.7 % AR after 62 d (concentration level of 100 µg/L). The maximum levels of the metabolite eluting after 25.8 min were 26.1 AR (concentration level of 10 µg/L) and 20.0 % AR (concentration level of 100 µg/L), in both cases observed after 42 d.
In addition, two transformation products with a retention of 18.7 min and 27.2 min were found at levels > 5 % AR in at least one of the incubation concentrations.

Identification of the metabolites detected by radio-HPLC was performed by LC-HR-MS analysis of representative extracts. Two strategies were applied in order to deliver as much information on the observed degradation products as possible. The first approach was the search at retention time range of the metabolites for molecular ion masses with an isotopic pattern comprising one or two 14C atoms besides the molecular ion without 14C label. The identity of most unknown signals at amounts above 1% AR could be determined.
However, at low amounts signals originating from metabolite molecules comprising a 14C labelled may not be detectable by LC-HR-MS. Therefore, in a second approach, the data collected by HR-MS was searched for molecules predicted as potential degradation products of dimethyldiphenylether (DTE) isomers by EAWAG-BBD Pathway Prediction Model. However, no additional metabolites were identified by this procedure.

The metabolites tolyloxy-phenylbenzoicacid (“DTE monocarboxylic acid”) and dicarboxydiphenyl ether (“DTE dicarboxylic acid”)were the most significant degradation products and were detected as the most at different retention times. Considering the retention times observed with radio-HPLC analysis,the metabolites were monitored in three resp. two peaks indicating that they consist of a mixture of isomers. The applied method HPLC-HRMS cannot yield distinct structural information on isomers. Furthermore all degradation products exceeding 10% AR were either tolyloxy-phenylbenzoicacid or dicarboxydiphenyl ether.

In extracts of sterilised samples no degradation of dimethyldiphenylether isomers was observed. The detected amounts of the single isomers, were corresponding to their initial amounts. This result demonstrates that the degradation of the dimethyldiphenylether isomers was a microbial process.

Although present at amounts above 1% AR, the metabolites eluting in at Rt 20.2 and 20.9 (Unknown Rt 20.2 and 20.9) in the original radio-HPLC analysis could not be identified. At the retention time range of these metabolites no significant MS signals were detected. Considering this result, it is obvious, that the unknown metabolites are not or only poorly ionised and cannot be detected by MS at such low concentrations. Based on their chromatographic behaviour, the polarity of the unknown metabolites is between the polarity of the methylphenoxybenzoic acid and dicarboxydiphenyl ether.

Evaporation of parent compound:
yes
Volatile metabolites:
yes
Residues:
no
Details on results:
RECOVERY
The overall recoveries ranged between 90 and 110 % of initially applied radioactivity for all samples up to incubation day 42. For the later sampling time points the recoveries were below 90%. For samples applied at the concentration level 10 µg/L the recoveries were all in the range of 70.7% and 77.5% for the samples taken between 62d and 92d. %. For samples applied at the concentration level 100 µg/L the recoveries were better and in the range of 82.9% and 94.5% for the samples taken at 62d and 76d, only in the final sampling time the recoveries decreased below 80% and were 78.3% and 72.9%. It is assumed that the formed volatiles, e.g. 14CO2 could not be trapped completely during sample preparation or were lost during extraction procedure and that this is the reason for the reduced recoveries. Alternatively, an adsorption of degradation products to surfaces during sample work-up could have occurred.
After the first significant decrease of the mass balance was observed for samples of 10 µg/L concentration level taken at day 62, additional attempts were made to recover the missing radioactivity for these samples. The solid residue after extraction, consisting of the amended sediment and the biosolids generated during the incubation, was further extracted using more polar extraction agents (methanol, methanol+trifluoroacetic acid, methanol+ammonia) and subsequently combusted. Furthermore the rinsed adsorber material of the Tenax traps was also combusted. In result further 3-5% (10 µg/L concentration level) and approx. 1% (100 µg/L concentration level) could be recovered, but especially for the 10 µg/L concentration level the recovery was still significantly <90%.

VOLATILES
In the rinsing solutions of the Tenax® traps only negligible amounts of radioactivity < 1 % AR were found. Thus, only small amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants were present in the headspace of the closed test system at sampling time point (< 1 % AR).
In the sodium hydroxide traps only small amounts of radioactivity (< 5 % AR) were detected. Significant amounts of radioactivity in the sodium hydroxide traps were observed at the end of incubation time. The maximal amounts were 3.6% (mean value) at concentration level of 10 µg/L after 76d of incubation and 1.7% (mean value) at concentration level of 100 µg/L after 92d of incubation. In the sodium hydroxide traps of the sterile samples no radioactivity was detected after 63d of incubation. The results show that mineralization is not the major degradation route but it is clearly associated with microbial activity.


EXTRACTABLE RADIOACTIVITY
The amount of radioactivity extracted from the surface water and the suspended sediment was constantly >90% up to incubation day 42 for both concentration levels. Afterwards a decrease was observed to 62% and 68% for the 10 µg/L and 100 µg/L concentration level until the end of incubation (92d). In sterilised surface water samples the extractable radioactivity was >90% AR for both concentration levels at the end of incubation period (63d). The different amounts of extractable radioactivity in biotic and abiotic samples also indicate degradation of the test item associated with microbial activity.

AQUEOUS PHASE AFTER FIRST EXTRACTION STEP
Generally, the amount of radioactivity remaining in the water phase after the partitioning increased from 0% AR at day 0 to values of approx. 9.0 % (concentration level of 10 µg/L) and 6 % AR (concentration level of 100 µg/L) until the end of incubation. In sterile surface water amounts of radioactivity <1% AR were found in the aqueous phase after the partitioning.
Results with reference substance:
The microbial activity of the test water used was determined by studying aerobic mineralization of 14C-sodium benzoate as an aerobically easy to degrade reference substance. It was however found, that the degradation was significantly slowed down under the test conditions (12 ± 2 °C, closed system), compared to standard test conditions (20 ± 2 °C, flow-through system). After 2 weeks 35% (mean value of four replicates) of the reference compound was degraded. A mineralization of 62.8 % AR of the reference substance was found within the incubation time of 60 days. A closed test setup had to be used due to high volatility of the test compound (Henry´s law constant 4.9 Pa m³/mol). The determined degradation rates of the reference compound indicate that the test system which had to be applied represents over-conservative experimental conditions yielding significantly slowed degradation not only of the reference substance but probably also of the test item.
At the end of incubation time mean mass balance for closed setup stayed in the recovery range of 90 -110 % of applied radioactivity with of 94.9 % and for flow-through setup slightly decreased to 89.5 % AR. For both incubation systems - flow-through vs closed setups, the microbial activity was comparable high with over 50 % mineralization of applied sodium benzoate within 7 day incubation at 12 °C.
In these later experiments the mineralisation was quite fast and system is much faster and no difference between the flow-through and the closed incubation system was observed. Therefore, the reason for the quite significant differences of the results is supposed to be the actual microbial population in the surface water at sampling time point. However, a direct correlation between the degradation of the reference compound sodium 14C-benzoate and the test item is not proved by the performed experiments.

HPLC based calculations:

SFO                  
 Isomer     Concentration Level     chi2  r2  Prob > t  DT50  DT90
 (%)  (-)  kdeg  (d)  (d)
 2,2´-Dimethyl-diphenylether        10 µg/L  11.2  0.5878  1.42*10-3  45.1  150
 100 µg/L  5.81  0.6147  1.26*10-4  65.0  216
 geometric mean        54.1  
 2,3´-Dimethyl-diphenylether         10 µg/L 7.16   0.9706  5.93 *10 -10  20.5  68
 100 µg/L  11.6  0.8664  4.93 *10 -7  32.9  109
 geometric mean        26.0  
 2,4´-Dimethyl-diphenylether       10 µg/L   10.3  0.8758

 2.13 *10 -6

 28.4

 94.5

 100 µg/L  10.2  0.9273  5.9 *10-9  29.9  99.3
 geometric mean        29.1  
 3,3´-Dimethyl-diphenylether        10 µg/L  6.37  0.9712   4.36*10 -9 14.9   49.6
 100 µg/L  11.4  0.931  8.85 *10 -9  26.9  89.2
 geometric mean        20.0  
 3,4´-Dimethyl-diphenylether        10 µg/L  4.78  0.9850  7.4*10 -12 15.7   52.3
 100 µg/L  12.3  0.9500  3.94 *10 -9  22.9  76
 geometric mean        19.0  
 4,4´-Dimethyl-diphenylether       10 µg/L   6.01  0.8281  4.44 *10 -5 17.7   58.9
 100 µg/L  12.1  0.9118  6.78 *10 -7 21.9  72.6
 geometric mean        19.7  

GC-MS based calculations:

SFO                  
 Isomer     Concentration Level     chi2  r2  Prob > t  DT50  DT90
 (%)  (-)  kdeg  (d)  (d)
 2,2´-Dimethyl-diphenylether        10 µg/L  10.6

 0.6692

 2.43*10 -4

 55.1

 183

 100 µg/L

7.85

 0.5914

 1.82*10-4

 72.1

 240

 geometric mean

 

 

 

 63.0

 

 2,3´-Dimethyl-diphenylether      

  10 µg/L

10.9 

 0.8907

 3.75 *10 -6

 19.2

 63.9

 100 µg/L

 11.1

 0.8162

 3.93 *10 -6

 33.1

 110

 geometric mean

 

 

 

 25.2

 

 2,4´-Dimethyl-diphenylether      

10 µg/L 

 8.28

 0.9009

 1.37 *10 -6

 20.9

 69.5

 100 µg/L

 8.87

 0.8976

 1.35 *10 -7

 28.0

 92.9

 geometric mean

 

 

 

 24.2

 

 3,3´-Dimethyl-diphenylether      

 10 µg/L

 11.6

 0.9049

  2.31*10 -6

16.8 

 55.8

 100 µg/L

 9.87

 0.8692

 8.75 *10-7

 27.4

 91.0

 geometric mean

 

 

 

 21.5

 

 3,4´-Dimethyl-diphenylether      

 10 µg/L

 10.8

 0.9027

 2.05*10 -6

16.8 

 55.8

 100 µg/L

 9.24

 0.9084

 1.57 *10 -7

 21.9

 72.7

 geometric mean

 

 

 

 21.5

 

 4,4´-Dimethyl-diphenylether      

10 µg/L 

 10.2

 0.8263

 1.99 *10-5

26.7 

 88.8

 100 µg/L

 9.82

 0.8486

 1.98 *10 -5

19.4

 64.3

 geometric mean

 

 

 

 22.8

 

Distribution of radioactivity in surface water treated with dimethyldiphenylether isomer mixture at a concentration of 10 µg/L and incubated at 12°C in % of applied radioactivity (% AR) in the extracts, the aqueous phases, traps, rinsing solutions and the overall recovery.

    % of Applied Radioactivity by Incubation time (days)  
   Rep  0  3  7  14  21  27  42  62  76  92  Sterile 63
 Combined extracts        1  104.3  101.3  99.8  101.8  97.7  93.9  93.8  69.8  64.1  60.9  86.7
 2  105.8  104.0  102.0  101.8  97.0  90.8  96.9  64.0  62.4  63.2  107.3
 Mean  105.1  102.7  100.9  101.8  97.4  92.4  95.3  66.9  63.3  62.0  97.0
 Water after extraction        1  0.0  0.0  0.0  0.5  1.0  0.0  2.5  4.1  10.8  8.4  0.2
 2  0.0  0.0  0.0  0.5  1.3  0.1  2.1  7.7  7.3  8.8  0.4
 Mean  0.0  0.0  0.0  0.5  1.2  0.0  2.3  5.9  9.0  8.6  0.3
 Tenax Extract        1  0.0  0.2  0.6  0.2  0.2  0.0  0.20.0  0.1  0.1  0.0  0.5
 2  0.0  0.2  0.3  0.3  0.1  0.0  0.2  0.0  0.1  0.0  0.8
 Mean  0.0  0.2  0.5  0.3  0.2  0.0  0.2  0.0  0.1  0.0  0.6
 Alkaline Traps (NaOH)        1  n.d  0.0  0.0  0.0  0.1  0.1  0.3  0.0  2.5  1.4  0.0
 2  n.d.  0.0  0.0  0.1  0.1  0.1  0.1  1.1  4.7  2.0  0.0
 Mean  n.d.  0.0  0.0  0.0  0.1  0.1  0.2  0.6  3.6  1.7  0.0
 TOTAL        1  104.3  101.5  100.4  102.6  99.0  94.0  96.7  73.9  77.5  70.7  87.4
 2  105.8  104.2  102.3  102.7  98.6  91.0  99.3  72.8  74.5  73.9  108.5
 Mean  105.1  102.9  101.4  102.7  98.8  92.5  98.0  73.3  76.0  72.3  97.9
 Overall mean    +-
StdDev
     92.3  +-  13.2          

n.d. = not determined

Distribution of radioactivity in surface water treated with dimethyldiphenylether isomer mixture at a concentration of 100 µg/L and incubated at 12°C in % of applied radioactivity (% AR) in the extracts, the aqueous phases, traps, rinsing solutions and the overall recovery.

     % of Applied Radioactivity by Incubation time (days)                              
   Rep  0  3  7  14  21  27  42  62  76  92  Sterile 63
 Combined extracts        1  101.7 99.7  98.2  97.1  92.3  90.2  90.7  88.5  89.9  70.8  105.2
 2  99.6  98.8  96.0  93.6*  92.8  91.8  90.5  79.4  80.2  65.1  105.3
 Mean  100.7  99.3  97.1  95.4  92.6  91.0  90.6  83.9  85.0  68.0  105.2
 Water after extraction        1  0.0  0.0  0.1  0.2  0.5  0.7  1.6  2.6  3.8  5.2  0.2
 2  0.0  0.0  0.1  0.2  0.6  1.2  1.7  3.0  3.5  6.8  0.3
 Mean  0.0  0.0  0.1  0.2  0.6  1.0  1.7  2.8  3.7  6.0  0.2
 Tenax extract        1  0.0  0.5  0.3  0.3  0.3  0.0  0.1  0.1  0.3  0.0  0.2
 2  0.0  0.3  0.2  0.2  0.2  0.0  0.1  0.0  0.0  0.0  0.2
 Mean  0.0  0.4  0.3  0.2  0.3  0.0  0.1  0.0  0.1  0.0  0.2
 Alkaline Traps (NaOH)       1  n.d.  0.0  0.0  0.0  0.0  0.0  0.1  0.1  0.5  2.3  0.0
 2  n.d.  0.0  0.0  0.0  0.0  0.0  0.2  0.5  1.4  1.0  0.0
 Mean  n.d.  0.0  0.0  0.0  0.0  0.0  0.2  0.3  1.0  1.7  0.0
 TOTAL        1  101.7  100.3  98.6  97.6  93.2  90.9  92.6  91.3  94.5  78.3  105.6
 2  99.6  99.1  96.4  94.0  93.6  93.0 92.5 82.9 85.1 72.9   105.9
 Mean  100.7  99.7  97.5  95.8  93.4  92.0  92.5  87.1  89.8  75.6  105.7
 Overall mean  +/- StdDev    92.4  +/-  7.3              

n.d.: not determined

* Including rinsing solution of the top piece of the incubation vessel.

Validity criteria:
mineralization of the reference substance
Observed value:
Mineralization was not finished after 7-14 days. The 50-60% level was reached after 30 days (12°C) and after 50 and 60 days 60-70% were reached.
Validity criteria fulfilled:
not applicable
Remarks:
The mineralization of the reference substance was much slower, compared to results observed in flow-through incubation systems. However, validity criteria are stated for standard flow-through conditions, applicability in the closed setup is unclear.
Validity criteria:
Recovery
Observed value:
The total recoveries ranged between 90 and 110% of applied radioactivity except for 10 replicates which showed recoveries in the range of 70.7% to 85.1%. It is however stated that these deficiencies do not have an impact on the validity of the study.
Validity criteria fulfilled:
yes
Remarks:
The total recoveries ranged between 90 and 110% of applied radioactivity except for 10 replicates in the range of 70.7% to 85.1%. Volatiles, e.g. 14CO2 could probably not be trapped completely during incubation and/or sample preparation.
Validity criteria:
chi2 SFO kinetic model fitting
Observed value:
chi2 < 15% for all values
Validity criteria fulfilled:
yes
Conclusions:
A study was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to investigate the aerobic mineralization of dimethyldiphenylether isomers according to the OECD-Guideline 309 " Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test".
Replicate samples of the test item dimethyldiphenylether isomer mixture were taken for analyses at 0, 3, 7, 14, 21, 27, 42, 62, 76 and 92 days after application. The surface water and the suspended sediment was partitioned with ethyl acetate and dichloromethane. Extracts were analysed for the test substance and possible degradation products by HPLC.

Based on the achieved data sets (radio-HPLC and GC-MS), the calculation of rate constants and DT50/DT90 values of the single dimenthyldiphenylether isomers in surface was calculated by means of the computer software “CAKE” version 3.3 (Release) running on R version 3.0.0 (2013-04-03)
A total radioactivity balance and the distribution of radioactivity in every subsample were established at each sampling day. The total recoveries ranged between 90 and 110% of applied radioactivity except. Volatile metabolites with low molecular weight or high Henry constants volatilizinge during the aerobic degradation in the closed test system (Tenax® traps) were always < 1 % AR.
The results show that mineralization of the test item during the aerobic incubation is not a major degradation route of dimethyldiphenylether isomers. Maximum amounts of 2 % AR were detected in the sodium hydroxide traps. In sterile samples after 63 days of incubation, radioactive amounts in the organic extract and aqueous phase were found in ranges which are comparable to the samples at test start.

In extracts of sterilised samples no degradation of dimethyldiphenylether isomers was observed. The detected amounts of the single isomers, were corresponding to their initial amounts. This result demonstrates that the degradation of the dimethyldiphenylether isomers was a microbial process.

The geometric mean DT50 values of dimethyldiphenylether isomers based on SFO kinetics are comparable between both analytical methods. With exception of one isomer, all values of other isomers are in the range of 19.0 to 29.1 days (based on data of HPLC analysis) and 19.2 to 25.2 (based on data of GC-MS analysis)

The geometric mean DT50 values of isomer 2,2´-dimethyldiphenylether are 54.1 and 63.0 days, based on HPLC and GC-MS analysis.
The GC/MS results are considered more reliable and are taken for the final calculation of the DT50 values. Nevertheless, also the results based on the HPLC method are reported here.

Executive summary:

A study was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to investigate the aerobic mineralization of dimethyldiphenylether isomers according to the OECD-Guideline 309 " Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test". The study was conducted in accordance with the principles of Good Laboratory Practice using 14C-labelled test item.

Dimethyldiphenylether isomers were incubated in test water amended with sediment using the test concentrations 10 µg/L and 100 µg/L for the isomer mixture under dark, aerobic conditions. The amount of the single isomers in the mixture was corresponding to the composition of the technical product. Sterile samples were additionally prepared to enable differentiation between biotic and abiotic degradation.

The mineralization of dimethyldiphenylether isomers was studied over an incubation time of 92 days at 12°C ± 2°C. Since the test substances are volatile, the test was conducted in a closed test setup.

The microbial activity of the test water used was determined by studying aerobic mineralization of 14C-sodium benzoate as an aerobically easy to degrade reference substance. It was however found, that the degradation was significantly slowed down under the test conditions (12 ± 2 °C, closed system), compared to standard test conditions (20 ± 2 °C, flow-through system). After 2 weeks 35% (mean value of four replicates) of the reference compound was degraded. A mineralization of 62.8 % AR of the reference substance was found within the incubation time of 60 days. A closed test setup had to be used due to high volatility of the test compound (Henry´s law constant 4.9 Pa m³/mol). The determined degradation rates of the reference compound indicate that the test system which had to be applied represents over-conservative experimental conditions yielding significantly slowed degradation not only of the reference substance but probably also of the test item. At the end of incubation time mean mass balance for closed setup stayed in the recovery range of 90 -110 % of applied radioactivity with of 94.9 % and for flow-through setup slightly decreased to 89.5 % AR. For both incubation systems - flow-through vs closed setups, the microbial activity was comparable high with over 50 % mineralization of applied sodium benzoate within 7 day incubation at 12 °C.

Replicate samples of the test item dimethyldiphenylether isomer mixture were taken for analyses at 0, 3, 7, 14, 21, 27, 42, 62, 76 and 92 days after application. The surface water and the suspended sediment was partitioned with ethyl acetate and dichloromethane. Extracts were analysed for the test substance and possible degradation products by HPLC.

A total radioactivity balance and the distribution of radioactivity in every subsample were established at each sampling day. The total recoveries ranged between 90 and 110% of applied radioactivity except for 6 replicates of the concentration level 10 µg/L and for 4 replicates of the higher concentration level of 100 µg/L. These replicates were all in the range of 70.7% and 85.1%. It is assumed that the formed volatiles, e.g. 14CO2 could not be trapped completely during incubation and/or sample preparation.

The amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants volatilizinge during the aerobic degradation in the closed test system (Tenax® traps) were always < 1 % AR.

The results show that mineralization of the test item during the aerobic incubation is not a major degradation route of dimethyldiphenylether isomers. Maximum amounts of 2 % AR were detected in the sodium hydroxide traps.

The amount of radioactivity extracted from the surface water by ethyl acetate decreased continuously in concentration levels from 105.7 % (concentration level 10 µg/L) and 100.7 % AR (concentration level 100 µg/L) at the beginning of incubation to 62.0 % (concentration level 10 µg/L) and 68.0 % AR (concentration level 100 µg/L) until the end of incubation. The amount of radioactivity remaining in the water phase after the partitioning with ethyl acetate increased to values of 8.6 % (concentration level of 10 µg/L) and 6.0 % AR (concentration level of 100 µg/L) until the end of incubation. In sterile samples after 63 days of incubation, radioactive amounts in the organic extract and aqueous phase were found in ranges which are comparable to the samples at test start.

In extracts of sterilised samples no degradation of dimethyldiphenylether isomers was observed. The detected amounts of the single isomers, were corresponding to their initial amounts. This result demonstrates that the degradation of the dimethyldiphenylether isomers was a microbial process.

The obtained data sets were analysed using the program CAKE version 1.3. The kinetic models considered for the analysis of dimethyldiphenylether isomers were SFO (Single First Order), DFOP (Double First Order in Parallel), HS (Hockey Stick), and FOMC (First Order Multi Compartment). According to the results the best fitting results were obtained when considering SFO kinetics.

The geometric mean DT50 values of dimethyldiphenylether isomers based on SFO kinetics are comparable between both analytical methods. With exception of one isomer, all values of other isomers are in the range of 19.0 to 29.1 days (based on data of HPLC analysis) and 19.2 to 25.2 (based on data of GC-MS analysis).

The individaul DT50 values of isomer 2,2´-dimethyldiphenylether are 54.1 and 72.1 days, with a geometric mean of 63 days based on GC-MS analysis.

The GC/MS results are considered more reliable and are taken for the final calculation of the DT50 values. Nevertheless, also the results based on the HPLC method are reported.

Based on the achieved data sets (radio-HPLC and GC-MS), the calculation of rate constants and DT50/DT90 values of the single dimenthyldiphenylether isomers in surface was calculated by means of the computer software “CAKE” version 3.3 (Release) running on R version 3.0.0 (2013-04-03).

The kinetics considered for all data were “single first order” (SFO), “First order multi compartment” (FOMC), "hockey stick (HS), and "Double first order in parallel" (DFOP) for the parent compound. SFO delivered the best fit kinetics for the parent compound.

Three metabolites have been identified (DTE mono carboxylic acid, DTE dicarboxylic acid and DTE Xanthenone. The primary step of degradation is oxidation of the hydroxy group.

Description of key information

In an OECD 309 study (Fraunhofer 2021) the aerobic mineralisation of dimethyldiphenylether isomer mixture is investigated under aerobic conditions according to the OECD-Guideline 309 using surface water from a natural lake and 14C-labelled test item.


 


The geometric mean DT50 values of dimethyldiphenylether isomers based on SFO kinetics are in the range of 19.2 to 25.2 (based on data of GC-MS analysis), with exception of one isomer, 2,2'-diphenylmethylether.


The individual DT50 values of isomer 2,2'-dimethyldiphenylether are within 55.1 and 72.1 days, based on GC-MS analysis. The geometric mean value is calculated as 63 days at 12°C.


Three metabolites have been identified (DTE mono carboxylic acid, DTE dicarboxylic acid and DTE Xanthenone. The primary step of degradation is oxidation of the hydroxy group.

Key value for chemical safety assessment

Half-life in freshwater:
63 d
at the temperature of:
12 °C

Additional information

An OECD 309 study monitoring all 6 isomers of DTE was performed based on a final decision of ECHA. The OECD 309 study is considered as the worst case biological system considering biodegradation due to the low concentration of degraders.As one of the DTE isomers (2,2'-DTE) was found to be vP, further testing in a water-sediment system or in soil are not required.