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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 - 29 Oct 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: ISO/DIS method 10708: BODIS test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Inoculum was obtained from the municipal WWTP Hochdahl, Germany
- Pretreatment: none
- Preparation of inoculum for exposure: pre-incubation with mineral medium for one week (at 18 - 22 °C)
- Concentration of sludge: 30 mg suspended solids/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to OECD guideline 301
- Test temperature: 20 - 25 °C
- Other: The vessels were shaken continously throughout the test.

TEST SYSTEM
- Culturing apparatus: 300 mL high breast bottles containing ~ 200 mL test solution
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: The bottles were aerated with compressed air by means of a sintered glass tube until oxygen saturation was reached.
- Measuring equipment: The COD was determined in a variant og ISO method 6060. Oxygen determinations were performed using a oxygen-electrode (WTW oximeter Oxi 2000).

SAMPLING
- Sampling frequency:every 7 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: no
- Abiotic sterile control: no
- Toxicity control: no
- Other: reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
100 mg/L as ThOD
Parameter:
% degradation (O2 consumption)
Value:
1
Sampling time:
28 d
Details on results:
Under the described test conditions only 1% of the test substance was degraded within the 28 day test period.
Parameter:
COD
Value:
2 529 other: mg O2/ g test substance (active matter)
Results with reference substance:
The reference substance showed biodegradation of 75% after 7 days and of 83% after 28 days incubation time.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance showed to be neither readily nor inherently biodegradble under the conditions of a BODIS test performed according to ISO/DIS guideline 10708.
Executive summary:

The test substance was tested for its biodegradability according to ISO guideline 10708 ( BOD adapted to insoluble test substances) and GLP using non-adapted activated sludge as inoculum . As almost no biodegradation was observed over 28 days (based on BO2 consumption), the test substance can be judged as not readily biodegradable.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented and peer reviewed publication which meets basic scientific principles
Principles of method if other than guideline:
BODIS test following "Standard methods for the Examination of water and wastewater" (Am. Pub. Health Assn.)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: settled domestic waste water and mixture of seawater/wastewater, resp., inoculum non-adapted
Details on inoculum:
Freshwater:
settled domestic wastewater (3 ml/bottle)
seawater
the seed used was developed in seawater taken from Lavaca Bay (Texas), the seed was mainted by adding small
amounts of settled raw wastewater every 3 to 4 days as a source of substrate, seed bacteria, and growth factors
Duration of test (contact time):
20 d
Initial conc.:
>= 3 - <= 10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to standard method

TEST SYSTEM
- Culturing apparatus: BOD bottles
- Number of culture flasks/concentration: duplicate bottles per test concentration; at least two test concentrations of three (3, 7, 10 mg/L)

SAMPLING
- Sampling frequency: day 0 and every 5 days

freshwater test: BOD bottles were half filled with aerated dilution water and settled domestic waste water (3 mL/bottle) was added
containing minerals and buffer; concentrations of TS: 3, 7, and 10 mg/l; at least two of the concentrations were tested
in duplicate; the bottles were reaerated, when the DO dropped below 4.0 mg/l

seawater test:
the biodegradation was carried out in synthetic seawater; concentration of TS: 3, 7, and 10 mg/l; at least two
concentrations were tested in duplicate; the bottles were reaerated, when the DO dropped below 4.0 mg/l
Parameter:
% degradation (O2 consumption)
Value:
96
Sampling time:
20 d
Remarks on result:
other: freshwater, non-adapted
Parameter:
% degradation (O2 consumption)
Value:
86
Sampling time:
20 d
Remarks on result:
other: salt water, non-adapted
Details on results:
Kinetic of test substance (in %):
fresh water:
= 90 % after 5 days
= 89 % after 10 days
= 87 % after 15 days
= 96 % after 20 days

salt water:
= 55 % after 5 day(s)
= 74 % after 10 day(s)
= 78 % after 15 day(s)
= 86 % after 20 day(s)
Validity criteria fulfilled:
not applicable
Interpretation of results:
other: biodegradable
Conclusions:
Degradation of phenol was tested in frewh water and salt water applying the published BOD procedure. Non-adapted inoculum was applied.
The results show > 70 % biodegradation for fresh and salt water within the first 10 days indicating ready biodgradability of phenol.

Executive summary:

Degradation of phenol was tested in fresh water and salt water applying the published BOD procedure. Test substance was added as sole carbon source. Degradation was followed by the oxygen decrease in the test mixture incubated in sealed test vessels. Non-adapted inoculum was applied. For the fresh water test settled and filtered domestic waste water was used as seed. For the seawater biodegradation test the seed was maintained by adding small amounts of settled raw wastewater about every 3 to 4 days to seawater taken from the environment.

Duplicate test vessels were analysed per test concentration and sampling time. The results show > 70 % biodegradation for fresh and salt water within the first 10 days indicating ready biodgradability of phenol.

Endpoint:
biodegradation in water: screening tests
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Validated QSAR model. Calculation for minor component 2-ethylhexanoic acid.
Principles of method if other than guideline:
Calculation based on BIOWIN v4.10, Estimation Programs Interface Suite™ for Microsoft® Windows v 4.10. US EPA, United States Environmental Protection Agency, Washington, DC, USA.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on results:
For detailed description on the model and its applicability, see "Any other information on materials and methods incl. tables".

 BIOWIN v4.10 Results

Biowin1 (Linear Model Prediction): Biodegrades Fast

Biowin2 (Non-Linear Model Prediction): Biodegrades Fast

Biowin3 (Ultimate Biodegradation Timeframe): Days-Weeks

Biowin4 (Primary Biodegradation Timeframe): Hours-Days

Biowin5 (MITI Linear Model Prediction): Biodegrades Fast

Biowin6 (MITI Non-Linear Model Prediction): Biodegrades Fast

Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

Ready Biodegradability Prediction: YES

TYPE

 NUM

Biowin1 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

Frag

 1

1 

Linear C4 terminal chain [CCC-CH3]

Aliphatic acid [-C(=O)-OH]    

 0.1084

0.0727

 0.1084

0.0727

MolWt

 * 

Molecular Weight Parameter               

        

-0.0687

Const

 * 

Equation Constant                        

        

0.7475

RESULT  

 

Biowin1 (Linear Biodeg Probability)    

 

0.8600

 

TYPE

 NUM

Biowin2 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

Frag

 1

1

Linear C4 terminal chain [CCC-CH3] 

Aliphatic acid [-C(=O)-OH]     

 1.8437

0.6431

 1.8437

0.6431

MolWt

 * 

Molecular Weight Parameter               

        

 -2.0479

RESULT  

 

Biowin2 (Non-Linear Biodeg Probability)  

 

 0.9692

 

TYPE

 NUM

Biowin3 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

Frag

 1

1

Linear C4 terminal chain [CCC-CH3] 

Aliphatic acid [-C(=O)-OH] 

 0.2983

0.3646

 0.2983

0.3646

MolWt

 * 

Molecular Weight Parameter               

        

 -0.3187

Const

 * 

Equation Constant                        

        

 3.1992

RESULT  

 

Biowin3 (Survey Model – Ultimate Biodeg) 

 

 3.5434

 

TYPE

 NUM

Biowin4 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

Frag

 1

1

Linear C4 terminal chain [CCC-CH3] 

Aliphatic acid [-C(=O)-OH]     

 0.2691

0.3856

 0.2691

0.3856

MolWt

 * 

Molecular Weight Parameter               

        

 -0.2081

Const

 * 

Equation Constant                        

        

 3.8477

RESULT  

  

Biowin4 (Survey Model - Primary Biodeg)  

 

 4.2943

 

TYPE

 NUM

Biowin5 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

Frag

 1

2 

Aliphatic acid [-C(=O)-OH]

Methyl [-CH3]                           

 0.1812

0.0004

 0.1812

0.0008

Frag

Frag

4

1 

-CH2- [linear]

-CH- [linear]                          

 0.0494 -0.0507

 0.1977

-0.0507

MolWt

 * 

Molecular Weight Parameter               

        

 -0.4290

Const

 * 

Equation Constant                        

        

 0.7121

RESULT  

 

Biowin5 (MITI Linear Biodeg Probability)        

 

 0.6121

 

TYPE

 NUM

Biowin6 FRAGMENT DESCRIPTION        

 COEFF 

 VALUE

Frag

Frag

 1

Aliphatic acid [-C(=O)-OH]

Methyl [-CH3]                           

 1.1346

0.0194

 1.1346

0.0389

Frag

Frag

 4

1 

-CH2- [linear]

-CH- [linear]                          

 0.4295 -0.0998

 1.7180 -0.0998

MolWt

 * 

Molecular Weight Parameter               

        

-4.1633

RESULT  

 

Biowin6 (MITI Non-Linear Biodeg Probability)     

 

 0.7602

 

Interpretation of results:
readily biodegradable
Executive summary:

QPRF: BIOWIN v4.10

1.

Substance

See “Test material identity”

2.

General information

 

2.1

Date of QPRF

See “Data Source (Reference)”

2.2

QPRF author and contact details

See “Data Source (Reference)”

3.

Prediction

3.1

Endpoint
(OECD Principle 1)

Endpoint

Biodegradability

Dependent variable

Biodegradability

3.2

Algorithm
(OECD Principle 2)

Model or submodel name

BIOWIN

Model version

v. 4.10

Reference to QMRF

QMRF: Estimation of Aerobic Biodegradability using BIOWIN v4.10 (EPI Suite v4.11): BIOWIN1 to BIOWIN6 and Ready Biodegradability Prediction

Predicted value (model result)

See “Results and discussion”

Input for prediction

- Chemical structure via CAS number or SMILES

Descriptor values

- Structure fragments

- Molecular weight

3.3

Applicability domain
(OECD principle 3)

Domains (Appendix D, On-Line BIOWIN User’s Guide):

1) Molecular weight

See below (Assessment of estimation domain)

2) Fragments:

See below (Assessment of estimation domain)

3.4

The uncertainty of the prediction
(OECD principle 4)

Parameter

BIOWIN model

1

2

3

4

5

6

Total correct

264 / 295

275 / 295

167 / 200

165 / 200

485 / 589

488 / 589

% correct, total

89.5

93.2

83.5

82.5

82.3

82.9

% correct, fast

97.3 (181 / 186)

97.3 (181 / 186)

93.5 (101 / 108)

84.9 (101 / 119)

79.1 (201 / 254)

80.3 (204 / 254)

3.5

The chemical mechanisms according to the model underpinning the predicted result
(OECD principle 5)

The chemical structure influences the biodegradability of the substance. Therefore, chemical fragments were selected having a potential effect on biodegradability. In order to be able to predict the biodegradability probability of substances without these specific fragments, the molecular weight was integrated into the models.

 

References

- US EPA (2012). On-Line BIOWIN User’s Guide.

 

AppendixD - Fragment Coefficients for Biodegradation Models         
BIOWIN1 and BIOWIN2: Linear / Non-Linear Biodegradability         
Fragment description Coefficient   Training set fragment count     No. of instances
of each bond
found for the
current substance
  Linear Non-linear Min Max No. of compounds in training set containing the fragment .
Linear C4 terminal chain [CCC-CH3] 0,10843 1,8437 - 3 44 1
Aliphatic acid [-C(=O)-OH] 0,07269 0,6431 - 4 33 1
Molecular Weight --- --- 31,06 697,7   in range
.
BIOWIN3 and BIOWIN4: Ultimate / Primary biodegradability .
Fragment description Coefficient   Training set fragment count     No. of instances
of each bond
found for the
current substance
  Ultimate Primary Min Max No. of compounds in training set containing the fragment .
Linear C4 terminal chain [CCC-CH3] 0,29834 0,26907 - 3 26 1
Aliphatic acid [-C(=O)-OH] 0,364605 0,38557 - 1 10 1
Molecular Weight --- --- 53,06 697,65 - in range
.
BIOWIN5 and BIOWIN6: MITI Biodegradability Coefficients (Linear / Non-Linear) .
Fragment description Coefficient   Training set fragment count     No. of instances
of each bond
found for the
current substance
  Linear Non-Linear Min Max No. of compounds in training set containing the fragment .
Aliphatic acid [-C(=O)-OH] 0,181163 1,13459688  - 2 22 1
Methyl [-CH3] 0,000411 0,01942827  - 9 295 2
-CH2- [linear] 0,049416 0,42949426  - 51 214 4
-CH- [linear] -0,050672 -0,09977022  - 4 63 1
Molecular Weight  --- --- 30,02 959,2 - in range
.
Fragment description Coefficient   Min Max No. of compounds in training set containing the fragment No. of instances
of each bond
found for the
current substance
Linear C4 terminal chain [CCC-CH3] -0,317727891 ---  - 3 41 1
Aliphatic acid [-C(=O)-OH] 0,186772405 --- - 3 16 1
Methyl [-CH3] -0,079572183 ---  - 4 86 2
-CH2- [linear] 0,025989832 --- - 44 67 13
-CH- [linear] -0,165850299 ---  - 2 17 1
-C=CH [alkenyl hydrogen] -0,073523308 ---  - 11 15 2
Molecular Weight --- --- 46,07 885,46   in range
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance Benzaldehyde, 2-hydroxy-5-nonyl, oxime, branched (CAS No. 174333-80-3). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
yes
Remarks:
the activated sludge was not fed during the holding period of maximum seven days, the test was run at 22 °C, the test water composition was slightly changed, biological oxygen demand was monitored, no test item specific analysis was performed
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: obtained from a wastewater treatment plant (ARA Ergolz Il, Füllinsdorf, Switzerland) treating predominantly domestic wastewater
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. During the holding period of three days prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge defined amounts were added to test water to obtain a final concentration of 100 mg dry material per liter.
- Concentration of sludge: 4 g solids/L
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to OECD guideline 301F
- Test temperature: 22 'C, maintained with a built-in thermostat and checked once per week.
- pH: 7.1 - 7.6
- Continuous darkness: yes
- Other: incubation under continuous stirring in a SAPROMAT D12

TEST SYSTEM
- Culturing apparatus: 500 mL Erlenmeyer flasks
- Number of culture flasks/concentration: 2

CONTROL AND BLANK SYSTEM
- Toxicity control: yes (1 replicate)
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
The biochemical oxygen demand (BOD) of the test substance in the test media was in the normal range found for the inoculum controls. Consequently, the test substance was not biodegradable under the test conditions within 28 days.
Results with reference substance:
In the procedure controls, the reference item (sodium benzoate) was degraded by an average of 79% and 84% by Exposure Day 7 and 14, respectively; thus, confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 86%.

In the toxicity control, containing both the test substance and the reference item sodium benzoate, the test substance had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 30 mg/L

Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
In a study according to OECD test guideline 302C under GLP the test substance was not inherently biodegradable.
Executive summary:

The test substance was tested for its biodegradability in an OECD 302C study using activated sludge. The study was performed under GLP. There was no inhibitory effect on the sludge at the test concentration of 30 mg/L. As no biodegradation was observed over 28 days (based on BOD), the test substance is not considered to be inherently biodegradable.

Endpoint:
biodegradation in water: screening tests
Type of information:
other: review article
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented and peer reviewed publication which meets basic scientific
Principles of method if other than guideline:
Compilation of degradation pathways for different nonylphenol isomers and discussion of factors influencing degradation
GLP compliance:
no

Factors influencing degradation of NP

Position and length of alkyl chain As described for cresols and other short-chain AP, the position of substitution of the phenol ring by the nonyl chain seems to be decisive with regard to the degradation of NP by sphingomonads. S. amiense displayed a marked preference for p-NP isomers (de Vries et al. 2001). In the case of Sphingomonas sp. TTNP3, a residual accumulation of the o-NP isomers was observed during the time-course of incubation (Tanghe et al. 1999b; Corvini et al. 2004c). These facts indicate a higher persistence of o-NP, although the low endocrine potential and the small proportion of o- NP in tNP reduce the significance of this finding. With respect to the ipso-hydroxylation mechanism occurring in sphingomonads, the presence of a hydroxyl group adjacent to the alkyl chain could inhibit the reaction for o-NP isomers by increasing the steric hindrance at C-2. In bacteria, the length of the AP alkyl chain seems to be a decisive criterion for the degradation pathway and the microorganisms involved. In pseudomonads, many reports have demonstrated the degradation of short linear alkyl chain AP (Ajithkumar et al. 2003; Hopper and Cottrell 2003; Jeong et al. 2003). In these cases, the degradation pathways involve either catechol derivatives (Jeong et al. 2003) or a quinone methide intermediate (Hopper and Cottrell 2003), and no mechanisms similar to ipso-substitution were found. The degradation of various AP derivatives was determined during an incubation period of 2 weeks with Sphingomonas sp. TTNP3 (Corvini et al. 2004b). Both isopropyl and tert-butylphenol were not degraded, whereas the concentration of tert-octylphenol decreased as in the separate study by Tanghe et al. (2000). Although isopropyl and tert-butylphenol each possess a highly branched α-C atom, which would allow for the production of secondary and tertiary carbocationic intermediates, no degradation by Sphingomonas sp. TTNP3 occurred. These results are also in agreement with the fact that propyl- and hexylphenol are not degraded by S. cloacae (Fujii et al. 2000a). These observations indicate that degradation of AP by sphingomonads, known for their capacity to degrade NP, requires substrates having an alkyl chain with a minimal length. Linking the position and length of alkyl chains to the biodegradability of AP by fungi is complicated by several factors, such as the necessity to differentiate between cometabolic biotransformations and AP utilization for growth, the various enzymes known to oxidize AP but showing different substrate preferences and catalytic mechanisms, and the intracellular enzymes acting on NP, which is yet unknown. A striking observation is that o-cresol did not support the growth of filamentous fungi (Hofrichter et al. 1995; Garcia-Pena et al. 2005) and enabled only delayed growth in yeast (Middelhoven and Spaaij 1997). This contrasts with the fungal growth observed on para- and meta-substituted short-chain AP (Hofrichter and Scheibner 1993; Jones et al. 1993, 1994; Middelhoven and Spaaij 1997; Middelhoven et al. 2000, 2004; Garcia-Pena et al. 2005). Phenol hydroxylase from yeast is able to act on all three cresol isomers (Neujahr and Kjellen 1978). Fungal vanillyl alcohol oxidases oxidize p-cresol and other parasubstituted C2–C7 AP (Fraaije et al. 1998; Furukawa et al. 1999; van den Heuvel et al. 1998, 2000), and mushroom tyrosinase was reported to act on p-cresol and parasubstituted C2–C4 AP (Yamada et al. 2005). Laccase was shown to oxidize p-cresol at higher rates than o-cresol and also converts para- and ortho-substituted AP with larger alkyl substituents including NP (Bollag et al. 1988; Garzillo et al. 1998; Kobayashi and Higashimura 2003; Xu 1996; Junghanns et al. 2005), whereas m-cresol oxidation was not reported. This suggests that in laccases, the position of an alkyl chain is a more decisive criterion for AP oxidation than its length.

Influence of the structure of NP alkyl chain on NP degradation

The influence of the alkyl chain structure of NP isomers was considered in studies with sphingomonads. It was shown that 4-n-NP was not metabolized as a single source of carbon by Sphingomonas sp. TTNP3 and S. xenophaga (Corvini et al. 2004b; Gabriel et al. 2005a). These a priori unexpected results can again be explained by the mechanism of type II ipso-substitution where a primary carbocationic intermediate from the linear alkyl chain is energetically unlikely. Although the simultaneous decrease of all tNP isomers and the production of many nonanols were indicative of an aspecific degradation of isomers of tNP in S. cloacae and Sphingomonas sp. TTNP3 (Tanghe et al. 1999b; Fujii et al. 2000b; Corvini et al. 2004c) higher consumption rates were found for 4-[1-ethyl-1,3-dimethylpentyl] phenol than for tNP in Sphingomonas sp. TTNP3. Furthermore, slight differences in the biodegradation of the different isomers of NP were reported in Sphingomonas sp. TTNP3 and S. xenophaga. In the case of Sphingomonas sp. TTNP3, differences in the dead-end metabolite trace pattern were reported. Benzenediol derivatives were found for a lot of NP isomers in intracellular extracts of Sphingomonas sp. TTNP3 grown on tNP, whereas alkyloxyphenols were not detected for all of these isomers (Corvini et al. 2005). Resting cell assays over short incubation periods demonstrated that despite single isomers of NP, all being degraded via hydroquinone, a comparison of the degradation rates showed that 4-[1,1,5-trimethylhexyl]-phenol is degraded more slowly than 4-[1-ethyl-1,3-dimethylpentyl]phenol and 4-[1-ethyl-1,4-dimethylpentyl]phenol. 4-[1-ethyl-1,3-dimethylpentyl] phenol and 4-[1-ethyl-1,4-dimethylpentyl]phenol possess the same substitution pattern at the quaternary α-C atom (methyl, pentyl, and ethyl), whereas 4-[1,1,5- trimethylhexyl]-phenol possesses two methyl and one hexyl substituents (Fig. 6). These results are in agreement with those obtained after long incubation periods with S. xenophaga. This strain degrades 4-[1-ethyl-1,4-dimethylpentyl] phenol slightly more rapidly than 4-[1,1,2,4- tetramethylpentyl]-phenol, which has a dimethylated α-C atom with a highly branched pentyl group, and much faster than 4-[1,1-dimethylheptyl]-phenol, which also has a dimethylated α-C atom but a longer unbranched chain (Gabriel et al. 2005a). Thus, the substitution pattern of the α-C and the branching pattern of the largest alkylic substituent of the α-carbon may influence the degradation rate of the various isomers of NP and govern the pattern of the metabolites produced consecutively in this type II ipso substitution. Concerning the isomerism of the alkyl chain, to date, no effects on the biodegradation rates of the various p-NP isomers were clearly shown in the environment. Studies with soil microbial communities suggest that the p-NP isomers of tNP were similarly degraded (Topp and Starratt 2000). These results are corroborated by a recent report describing the formation of nitro metabolites when tNP is added to sludge-amended soil (Telscher et al. 2005). Nevertheless, the lack of studies demonstrating, on the one hand, the preferential degradation in environmental samples and, on the other hand, the specific persistence of defined isomers may be related to the difficulties of performing high quality chromatographic analysis of such complex mixtures. Furthermore, considering the various pathways, for instance the alkyl-chain oxidation by fungi, the structure of the alkyl-chain could also play a decisive role in the degradation of NP. The fact that both diastereomers of the 4-[1-ethyl-1,3-dimethylpentyl]phenol isomer were degraded at equal rates indicates that the stereochemistry of the alkyl side-chain is not crucial for the metabolism of the diastereomers by Sphingomonas sp. TTNP3. Stereospecificity apparently does not play a decisive role for ipso-substitution degradation pathways, but it would be interesting to assess whether this also applies to pathways requiring, for example, the hydroxylation of alkyl chains. No study so far has addressed degradation of single isomers of tNP by fungal organisms. However, effects of nonyl chain branching on fungal degradation would be expected from the reported differences in tNP and 4-n-NP removal by whole fungal cultures and isolated enzymes. Cunninghamella, Fusarium, and Mucor species removed 4-n-NP more efficiently than tNP, whereas the opposite was observed for P. chrysosporium and T. versicolor (Dubroca et al. 2005). Pertinent literatures indicate alkyl chain oxidation of at least some isomers of tNP by intracellular fungal enzymes (Junghanns et al. 2005; Moeder et al. 2006b), but the exact structures of nonyl chain branched tNP isomers that are susceptible to alkyl chain activation through intracellular enzymes and further breakdown remain to be elucidated. Otherwise, β-oxidation is thought to be restricted to linear or at least not highly branched alkyl chains (Osburn and Benedict 1966; van Ginkel 1996; Tanghe et al. 1999a,b). More insights into such processes are expected from studies applying the single nonyl chainbranched isomers contained in tNP, which are now available (Ruß et al. 2005). Laccase from the aquatic isolate UHH 1-6-18-4 oxidized tNP faster than 4-n-NP. This is in contrast to laccase from the aquatic hyphomycete C. aquatica, which converted 4-n-NP more efficiently than tNP (Junghanns et al. 2005). Laccases act on the phenolic OH group and therefore, branching of the nonyl chains located in the p-position should be less important for laccases than for enzymes directly attacking the alkyl chain or adjacent positions of the aromatic ring. Little steric hindrance of bulky p-substituents was shown for the laccase-catalyzed oxidation of phenols (Xu 1996).

Other factors governing the degradation of NP Because high NP concentrations can be toxic to microorganisms, the persistence of NP and other micropollutants may result from their trace concentrations in the environment (Kollmann et al. 2003). The growth and survival of cometabolic NP degraders independent from NP could be advantageous for NP removal when concentrations are well below the Km or Ks of the degrading enzymatic systems or specialist microorganisms. Enzyme affinity for xenobiotics is often low. For NP oxidation catalyzed by a laccase from a soil-derived ascomycete, a Km value of 5 mM (1.1 g l−1) was reported (Saito et al. 2003). Such high Km values would be rather unfavorable for an efficient removal of NP at the concentrations commonly found in water, which are found up to the microgram per liter range (Ying et al. 2002). Nevertheless, laccases from other organisms may differ in their kinetic features. Higher volumetric rates of 4- [1-ethyl-1,3-dimethylpentyl]-phenol degradation were reported in Sphingomonas sp. TTNP3 when the NP isomer was supplied above the hydrosolubility limit (25 μM) (Corvini et al. 2006a). For whole cells of this miroorganism, apparent Km and Vmax values were 230 μM and 0.050 μmol min−1 mg−1, respectively. Nevertheless, immobilized cells of S. cloacae were able to degrade NP in trace amounts, i.e., below 40 μg l−1 (Fujii et al. 2003). This high performance was attributed to the sorption of NP to the hydrophobic immobilization material leading to local higher concentrations in the vicinity of the bacteria. Similarly, kinetic limitations of laccase activity caused by low NP concentrations in aqueous systems may be diminished in sewage sludge and also in sediments where mean concentrations of up to approximately 1.5 g kg−1 and 2.1 mg kg−1, respectively, were reported (Pryor et al. 2002; Ying et al. 2003). The low biodegradability of NP may be explained by its strong sorption to, e.g., sediments and humic acids. This, in turn, should drastically decrease its bioavailability (Wheeler et al. 1997; Tanghe et al. 1998; Vinken et al. 2004). However, despite the high sorption coefficient of NP on humic acids, the degradation rates of NP in an axenic culture of Sphingomonas sp. TTNP3 were not affected (Vinken et al. 2004). Another study demonstrated that 4-n-NP was degraded within 40 days of incubation with organic material containing sediments, whereas the branched OP remained undegraded despite the higher sorption coefficient of 4-n-NP than that of 4-tert-OP (KOC 38,900 and 18,200, respectively) (Ying et al. 2003). In other words, the structure of alkyl chain prevailed over the effects of sorption. In the presence of natural organic matter, enzymatic coupling reactions catalyzed by fungal lignin-modifying enzymes may also contribute to humification processes through the formation of stable bound residues (Sarkar et al. 1988; Park et al. 2000; Ahn et al. 2002; Dec et al. 2003). Hydrosolubility of NP is increased at alkaline pH values when NP is deprotonated (pKa estimated between 10 and 12) and becomes less sorbed to organic materials and thus potentially more bioavailable (Ivashechkin et al. 2004). Nevertheless, extreme pH values were demonstrated to exert a negative effect on NP degradation by S. cloacae and very probably on its growth (Fujii et al. 2003). NP was not degraded at pH 9, while pH 6 or 8 led to an almost complete removal of 200 ppb NP. Among the critical parameters for the biodegradation of NP, oxygen appears to be particularly important. Anoxic periods led to a decrease in NP elimination in activated sludge and NP was more rapidly mineralized when oxygen diffusion into the sludge particles was facilitated in sludge– soil systems (Tanghe et al. 1998; Hesselsøe et al. 2001). Furthermore, the NP concentration profile increased with depth in anaerobic sediment layers (Espadaler et al. 1997). Few data concerning the biodegradation of NP in anaerobic environments are available. It was demonstrated that AP are not degraded during methanogenic treatment of sludge (Razo-Flores et al. 1996). However, the degradation of NP in sediments and in sewage sludge under anaerobic conditions was recently reported (Chang et al. 2004b, 2005a). Sulfate-reducing bacteria would be the major microbial component for the anaerobic degradation of NP in both environments, but the methanogen and eubacterial populations would also be involved. Nevertheless, doubts about the use of 4-n-NP remain because in the NP1EO (monoethoxylated tNP) degradation tests under the same incubation conditions, NP was accumulated and no information was provided concerning its further degradation. The easy degradation of linear alkane such as linear alkylbenzene sulfonate under anaerobic conditions would provide a rational explanation of these isolated cases of anaerobic degradation of NP (Mogensen et al. 2003). With regard to temperature, the data are quite heterogeneous. Decreasing temperature from 28 to 10 °C was shown to affect NP degradation bymicroflora in activated sludge (Tanghe et al. 1998). Nevertheless, inoculation of bioreactors with enriched bacterial consortia isolated from Swedish contaminated soil displayed satisfactory degradation rates for temperatures ranging from 15 down to 5.5 °C (Soares et al. 2005b). Further experiments were carried out with cold-adapted bacteria isolated from treatment plant in Sweden (Soares et al. 2003a). P. veronii was able to grow at temperatures near to 0 °C and it was able to degrade NP at 14 °C. On the whole, the results depend on the type of bacteria and the capability of extremophiles to carry out such reactions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented and peer reviewed publication which meets basic scientific principles
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Method of cultivation: semi continously cultured by the MITI method with a synthetic sewage
- Pretreatment: no
Duration of test (contact time):
10 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: K2HPO4 25.75 g/L; KH2PO4 8.5 g/L; Na2HPO4 * 12 H2O 44.6 g/L; NH4Cl 1.7 g/L; MgSO4 * 7 H2O 22.5 g/L; CaCl2 27.5 g/L; FeCl3 * 6 H2O 0.25 g/L
- sludge: 30 mg/L; the sludge for the seeding had been semiconituously cultured by the MITI method with a sysnthetic sewage of the following compostion: Glucose 25 g/L; Peptone 25 g/L; corn steep liquor 16 mL/L; KH2PO4 25 g/L; NaOH 16 g/L
- Temperature: 20 °C

TEST SYSTEM
- Measuring equipment: electrolytic respirometer
Key result
Parameter:
% degradation (O2 consumption)
Value:
62
Sampling time:
100 h
Details on results:
phenol concentration t1 (lag phase) k t[h] (degradation time) BOD/ThOD DOCd/DOC0
(mg/l) (h) (h-1) (h)
100 20 0.028 100 0.62 0.09
30 15 0.037 90 0.70 0.06
10 15 0.041 90 0.60 0.07
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
After a test period of 100 h, phenol (100 mg(L) resulted in a degradation rate of 62 %. The lag-phase was 20 h. Therefore, phenol is classified as readily biodegradable.
Executive summary:

The biodegradability of phenol (100 mg/L) was investigated in a ready biodegradability test (MITI). Activated sludge cultured in the laboratory was applied. Phenol was degegraded to 62 % within 100 h. The substance can be classified as readily biodegradable.

Description of key information

Not readily biodegradable: 1% biodegradation in 28 days test period (ISO 10708, BODIS test)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One test investigating the ready biodegradability of Ethanone, 1-(2-hydroxy-5-nonylphenyl)-, oxime, branched (CAS

244235-47-0) is available. The test was performed according to GLP and ISO guideline 10708 (BOD adapted to insoluble test substances) using non-adapted activated sludge as inoculum (Richterich 1996). Only 1% biodegradation was observed in a test period of 28 days (based on oxygen consumption). Thus, the test substance is not readily biodegradable.

Since no additional study assessing the inherent biodegradability of Ethanone, 1-(2-hydroxy-5-nonylphenyl)-, oxime, branched (CAS 244235-47-0) is available, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 Grouping of substances, a read-across to the structurally related source substance Benzaldehyde, 2-hydroxy-5-nonyl, oxime, branched (CAS 174333-80-3) was conducted. The only structural difference between the source substance and the target substance is the lack of a methyl group at the oxime carbon of the source substance. The read across is justified due to (i) the similarity of structure and functional groups and accordingly (ii) similar physico-chemical properties resulting in a similar environmental fate and ecotoxicity profile (see table below).

Table 1: Physico-chemical properties and ecotoxicological profile of the target and the source substance

Substance

Ethanone, 1-(2-hydroxy-5-nonylphenyl)-, oxime, branched

Benzaldehyde, 2-hydroxy-5-nonyl, oxime, branched

CAS number

244235-47-0

174333-80-3

Structure

see attachment

see attachment

Molecular formula

C17H27NO2

C16O2NH25

Molecular weight

~ 277 g/mole

~ 263 g/mole

PC parameter

 

 

Water solubility

> 0.02 < 1 mg/L (EU method A.6)

0.4 mg/L (EU method A.6)

Partition coefficient

> 5.7 (EU method A.8)

5.5 (EU method A.8)

Vapour pressure

< 1.5 Pa at 20 °C (OECD 104)

0.37 Pa at 20 °C (OECD 104)

Environmental fate

 

 

Biodegradability

1 % in 28 days (BODIS)

0 % in 28 days (OECD 302c)

Adsorption [log KOC]

3.9 (OECD 121)

3.7 (OECD 121)

Hydrolysis

not relevant

Ecotoxicology

 

 

Short-term toxicity to fish

[96h-LC50]

0.46 mg/L (EU method C.1)

1.1 mg/L (EU method C.1)

Short-term toxicity to aquatic invertebrates

[48h-EC50]

9.55 mg/L (OECD 202)

2.7 mg/L (EU method C.2)

Long-term toxicity to aquatic invertebrates

[21d-NOEC]

2.8 mg/L (OECD 211)

0.189 mg/L (OECD 211)

Short-term toxicity to algae

[72h-EC50]

760 mg/L (OECD 201)

36.3 mg/L (OECD 201)

Long-term toxicity to algae

[72h-NOEC]

472 mg/L (OECD 201)

14.9 mg/L (OECD 201)

Toxicity to microorganisms

[3h-EC10]

260.1mg/L (OECD 209)

200.4 mg/L (OECD 209)

The inherent biodegradation study with the source substance was performed according to GLP and OECD guideline 302C using non-adapted activated sludge as inoculum (Seyfried 2007). The biochemical oxygen demand (BOD) of the test substance in the test media was in the normal range found for the inoculum controls. Consequently, the test substance was not inherently biodegradable under the test conditions within 28 days. Based on the high structural similarity, which leads to similar behavior in the environment (as explained above), this conclusion can also considered to be true for Ethanone, 1-(2-hydroxy-5-nonylphenyl)-, oxime, branched (CAS 244235-47-0).

In addition, several screening tests for 4-nonylphenol are available (see table 2). In general, these tests show that 4-nonylphenol is not readily biodegradable according to OECD criteria (MITI 2002, Huels 1996 a,b). Degradation is observed if adapted inoculum was used (Staples et al. 1999, 2001) indicating 4-nonylphenol to be inherently biodegradable. However, the degradation potential is depending on several factors, such as the branching of the alkyl chain (see Corvini et al. 2006).

Table 2: Biodegradation test results for 4-nonylphenol*

Test

Result

Reliability

Reference

OECD 301C

0% after 14 days

2

MITI 2002

OECD 301B

47.5% after 28 days (adapted inoculum)

2

Staples et al. 2001

OECD 301F

57.4 – 68.4% after 28 days (adapted inoculum)

2

Staples et al. 1999

OECD 301B

0 % after 32 days (with and without emulsifier)

with adopted inoculum (adaption time 7 weeks):

0 % after 40 days (without emulsifier)

78 % after 40 days (with emulsifier)

4

(EU RAR 2002, Huels 1996a; Huels 1996b)

* Data obtained from the respective dossier published on the ECHA data base and from EU RAR 2002.

The two minor constituents phenol (CAS 108-95-2) and 2-ethylhexanoic acid (CAS 149-57-5) showed to be readily biodegradable based on available test data or valid QSAR calculations.

References

MITI 2002. Biodegradation and Bioconcentration of Exisiting Chemical Substances under the Chemical Substances Control Law. Information on the chemical published in the Official Bulletin of Economy, Trade and Industry.

Staples CA, et al. 2001. Ultimate biodegradation of alkylphenol ethoxylate surfactants and their biodegradation intermediates. Environ Toxicol Chem 20(11): 2450–2455

Staples CA, et al. 1999. Measuring the biodegradability of nonylphenol ether carboxylates, octylphenol ether carboxylates, and nonylphenol. Chemosphere 38(9): 2029-2039

EU RAR.2002. European Union Risk Assessment Report 4-nonylphenol (branched) and nonylphenol.

Huels. 1996a. Determination of the biological degradability of nonylphenol in the modified sturm test (EEC Directive 79/831 ENV/283/80) Report ST-3/84

Huels. 1996b. Determination of the biological degradability of nonylphenol in the modified sturm test (EEC Directive 79/831 ENV/283/80) Report ST-3a/84.