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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source
Justification for type of information:
According to the ECHA Practical Guide "How to use and report (Q)SARs 3.1", results of (Q)SARs may be used instead of testing when the conditions set in REACH Annex XI (1.3) are met:
(1) a (Q)SAR model where the scientific validity has been established should be used;
(2) the substance should fall within the applicability domain of the (Q)SAR model;
(3) the prediction should be fit for the regulatory purpose; and
(4) the information should be well documented.

1. SOFTWARE : EPI Suite (US EPA)

2. MODEL (incl. version number) : BIOWIN v4.10 ( Biowin1, Biowin2, Biowin3, Biowin4, Biowin5, Biowin6 and Biowin7).

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL : SMILE notation: Oc(c(O)ccc1)c1O and CAS n.: 87-66-1, CHEM : 1,2,3-Benzenetriol
MOL FOR: C6 H6 O3
MOL WT : 126.11

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
- Defined endpoint: the model must predict the same endpoint that would be measured to fulfil the requirements listed in REACH Annexes VII to X.
In the specific case, the end-point under investigation is "ready biodegradability".
The  Biodegradation  Probability  Program  (BIOWIN [1a, 1b]) used for the assessment estimates the probability of rapid aerobic biodegradation of  an organic chemical in the presence of mixed populations of environmental microorganisms.
[1a] US EPA. [2012 or insert current year]. Estimation Programs Interface Suite™ for Microsoft® Windows, v 4.11 or insert version used]. United States Environmental Protection Agency, Washington, DC, USA.
[1b] The  BIOWIN  program  was  developed  by Syracuse  Research  Corporation.  The prediction methodology was developed jointly by efforts of the Syracuse Research Corporation and the U.S. Environmental Protection Agency.

- Unambiguous algorithm: The algorithm underlying the model must be available to ensure transparency and reproducibility of the calculation.
The algorithm is well described in a Pavan et al. 2006 [2]
BIOWIN contains 7 separate models:
- Biowin1 =  linear probability model
- Biowin2 =  nonlinear probability model
- Biowin3 =  expert survey ultimate biodegradation model
- Biowin4 =  expert survey primary biodegradation model
- Biowin5 =  Japanese  MITI (Ministry of International Trade and Industry) linear model
- Biowin6 =  Japanese  MITI (Ministry of International Trade and Industry) nonlinear model
- Biowin7 = the newest model estimates anaerobic biodegradation potential.
Two independent training sets were used to develop four mathematical models for predicting aerobic biodegradability from chemical structure.
Two of the  models, based on linear and nonlinear regressions, calculate the probability of rapid biodegradation and can be  used to classify chemicals as rapidly or not rapidly biodegradable [2].

[2] Pavan M. and Worth A. P. Review of QSAR Models for Ready Biodegradation. 2006. EUR 22355 EN
.
- Defined domain of applicability:
The linear model (BIOWIN1) and the non-linear model (BIOWIN2) were developed using 264 chemicals.
BIOWIN3 and BIOWIN4 models are survey models because an expert panel was asked for their response to predicted rates for primary degradation (loss of parent chemical identity) and ultimate degradation (conversion to CO2 and H2O) under aerobic conditions.
The linear model (BIOWIN5) and non-linear model (BIOWIN6) for assessing organic compounds is based on the Japanese Ministry of International Trade and Industry (MITI) ready biodegradation test.
BIOWIN7 was developed using 169 chemicals and consists of a linear model and a non-linear model to assess the probability of rapid anaerobic biodegradation.

Biowin5 has 43 descriptors, and classifies a substance as either readily biodegradable (RB) or not readily biodegradable (NRB), based on the Japanese Ministry of International Trade and Industry (MITI) ready biodegradation test; i.e., OECD TG 301C.
The model was developed using multiple linear regression analysis of the binary dependent variable (0 for NRB or 1 for RB) against values for the 43 descriptors. The training set thus follows a discrete uniform distribution. The model estimates the likelihood that a chemical will degrade under the test conditions, with values of the dependent variable >= 0,5 being taken as predicting that the chemical will be RB in the 301C test. Some variables may be correlated. In the development of Biowin5, 884 discrete chemical substances (385 RB; 499 NRB) were randomly divided into training and validation sets (589 and 295 chemicals, respectively). The validation set was used to assess the predictive accuracy of Biowin5 in the present study. The training and validation chemicals used to develop Biowin5 (and 6) are liste [4].

Biowin3 estimates the time required to achieve complete ultimate biodegradation in a typical, or ‘evaluative’, aquatic environment. Aerobic ultimate biodegradation is the transformation of a parent compound to carbon dioxide and water, along with oxides of any other elements present in the test compound. The training set was obtained as follows. A total of 17 biodegradation experts reviewed a set of 200 molecular structures and were asked to estimate the amount of time required to achieve complete ultimate biodegradation in a typical aquatic environment. The experts were required to express their estimates in terms of prescribed words (days, weeks, months, etc.), to which integers were later assigned on a scale of 0 to 5. The numerical ratings obtained in this way from the completed surveys were averaged for each chemical.
[4] Boethling R.S. and Costanza J. Domain of EPI suite biotransformation models. SAR and QSAR in Environmental Research. 2010. 21 (5–6): 415–443

- Appropriate measures of goodness-of-fit and robustness and predictivity:
Two independent training sets were used to develop four mathematical models for predicting aerobic biodegradability from chemical structure.  All four of the models are based on multiple regressions against counts of 36 preselected chemical substructures plus molecular weight.  Two of the models, based on linear and nonlinear regressions, calculate the probability of rapid biodegradation and can be used to classify compounds as rapidly or not rapidly biodegradable.  The training set for these models consisted of qualitative summary evaluations of all available experimental data on biodegradability for 295 compounds.  The other two models allow semi-quantitative prediction of primary and ultimate biodegradation rates using multiple linear regression.  The training set for these models consisted of estimates of primary and ultimate biodegradation rates for 200 compounds, gathered in a survey of 17 biodegradation experts.  The two probability models correctly classified 90% of the compounds in their training set, whereas the two survey models calculated biodegradation rates for the survey compounds with R2 = 0.7.  These four models are intended for use in chemical screening and in setting priorities for further review.
- Mechanistic interpretation:
Biowin1 (Linear Model Prediction): Biodegrades Fast
Biowin2 (Non-Linear Model Prediction): Biodegrades Fast
Biowin3 (Ultimate Biodegradation Timeframe): Weeks
Biowin4 (Primary Biodegradation Timeframe): Days
Biowin5 (MITI Linear Model Prediction): Readily Degradable
Biowin6 (MITI Non-Linear Model Prediction): Readily Degradable
Biowin7 (Anaerobic Model Prediction): Biodegrades Fast
Ready Biodegradability Prediction: YES

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
- Descriptor domain: for each model, a description of the domain is reported below.
* Linear and Non-Linear Biodegradation Models (Biowin 1 and 2): The Appendix D (of the model) gives for each fragment the maximum number of instances of that fragment in any of the 295 training set compounds (the minimum number of instances is of course zero, since not all compounds had every fragment).  The minimum and maximum values for molecular weight are also given.  Currently there is no universally accepted definition of model domain.  However, users may wish to consider the possibility that biodegradability estimates are less accurate for compounds outside the MW range of the training set compounds, and/or that have more instances of a given fragment than the maximum for all training set compounds.  It is also possible that a compound may have a functional group(s) or other structural features not represented in the training set, and for which no fragment coefficient was developed; and that a compound has none of the 36 fragments in the model’s fragment library.  In the latter case, predictions are based on molecular weight alone. These points should be taken into consideration when interpreting model results.
* Ultimate and Primary Biodegradation Models (Biowin 3 and 4): The Appendix D (of the model) gives for each fragment the maximum number of instances of that fragment in any of the 200 training set compounds (the minimum number of instances is of course zero, since not all compounds had every fragment).  The minimum and maximum values for molecular weight are also given.  Currently there is no universally accepted definition of model domain.  However, users may wish to consider the possibility that biodegradability estimates are less accurate for compounds outside the MW range of the training set compounds, and/or that have more instances of a given fragment than the maximum for all training set compounds.  It is also possible that a compound may have a functional group(s) or other structural features not represented in the training set, and for which no fragment coefficient was developed; and that a compound has none of the 36 fragments in the model’s fragment library.  In the latter case, predictions are based on molecular weight alone.  These points should be taken into consideration when interpreting model results.
In addition to the above, the maximum and minimum values of the dependent variable (i.e. the averaged expert ratings) in the regressions, for both ultimate and primary degradation, may help characterize model domain.  They are:

Model                Min        Compound                        Max        Compound

Biowin3                1.44        pentabromoethylbenzene        3.89        ethylene glycol diacetate

Biowin4                2.37        pentabromoethylbenzene        4.57        ethylene glycol diacetate

* Linear and Non-Linear MITI Biodegradation Model (Biowin 5 and Biowin 6): The Appendix D (of the model) gives for each fragment the maximum number of instances of that fragment in any of the 589 training set compounds (the minimum number of instances is of course zero, since not all compounds had every fragment).  The minimum and maximum values for molecular weight are also given.  Currently there is no universally accepted definition of model domain.  However, users may wish to consider the possibility that biodegradability estimates are less accurate for compounds outside the MW range of the training set compounds, and/or that have more instances of a given fragment than the maximum for all training set compounds.  It is also possible that a compound may have a functional group(s) or other structural features not represented in the training set, and for which no fragment coefficient was developed; and that a compound has none of the fragments in the model’s fragment library.  In the latter case, predictions are based on molecular weight alone.  These points should be taken into consideration when interpreting model results.
* Anaerobic Biodegradation Model (Biowin 7): The Appendix D (of the model) gives for each fragment the maximum number of instances of that fragment in any of the 169 training set compounds (the minimum number of instances is of course zero, since not all compounds had every fragment).  The minimum and maximum values for molecular weight (not an independent variable) are also given.  Currently there is no universally accepted definition of model domain.  However, users may wish to consider the possibility that biodegradability estimates are less accurate for compounds outside the size (MW) range of the training set compounds, and/or that have more instances of a given fragment than the maximum for all training set compounds.  It is also possible that a compound may have a functional group(s) or other structural features not represented in the training set, and for which no fragment coefficient was developed; and that a compound has none of the 37 fragments in the model’s fragment library.  These points should be taken into consideration when interpreting model results.
- Structural and mechanistic domains:
In general, the Appendix D gives "Fragment Coefficients for Biodegradation Models" where each fragments of a chemical in linked to a specific coefficient. The (numerical) result obtained is compared to a threshold stated. According to this procedure, it was possible to predict the property (biodegradability).

* Ready Biodegradability Prediction:  (YES or NO)
The criteria for the YES or NO prediction are as follows:  If the Biowin3 (ultimate survey model) result is "weeks" or faster (i.e. days, days to weeks, or weeks) AND the Biowin5 (MITI linear model) probability is >= 0.5, then the prediction is YES (readily biodegradable).  If this condition is not satisfied, the prediction is NO (not readily biodegradable).
This method is based on the application of Bayesian analysis to ready biodegradation data for US Premanufacture Notification (PMN) chemicals, derived collectively from all six OECD301 test methods plus OECD310.  The approach is fully described in Boethling et al. (2004).
- Similarity with analogues in the training set:
- Other considerations (as appropriate): Boethling et al. compared the accuracy of different BIOWIN models (from BIOWIN1 to BIOWIN6) using a total of 944 PMN chemicals. The US EPA used 305 PMN chemicals, 439 chemicals from the MITI databases, and compared 200 PMN chemicals in the expert survey on which the BIOWIN3 and other BIOWIN models were evaluated. The greatest accuracy among BIOWIN models was demonstrated by BIOWIN3 (87%), whereas BIOWIN1 and BIOWIN2 models were deemed not suitable for estimating PMNs. developed the BIOWIN7 model using data from serum bottle tests (incubation period: at least 56 days) using 169 chemicals in the training set and 58 chemicals in two validation sets. The accuracy of the training set was approximately 90% and those of the validation sets were 77% and 91%, respectively [3].
[3] Seung L.J. EPI Suite: A Fascinate Predictive Tool for Estimating the Fates of Organic Contaminants. J Bioremediat Biodegrad 2016, 7:3.

6. ADEQUACY OF THE RESULT
[Explain how the prediction fits the purpose of classification and labelling and/or risk assessment]
Adequacy of the QSAR:
1) QSAR model is scientifically valid.
2) The substance falls within the applicability domain of the QSAR model.
3) The prediction is fit for regulatory purpose.
The prediction is adequate for the Classification and Labelling or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006: Annex XI Section 1.3


Qualifier:
according to
Guideline:
other: REACH Guidance on QSARs R.6
Deviations:
not applicable
GLP compliance:
no
Specific details on test material used for the study:
SMILE NOTATION: Oc(c(O)ccc1)c1O
CHEM : 1,2,3-Benzenetriol
MOL FOR: C6 H6 O3
MOL WT : 126.11
Oxygen conditions:
other: not relevant
Remarks:
a prediction was performed
Inoculum or test system:
other: not relevant
Remarks:
a prediction was performed
Details on inoculum:
not relevant
Details on study design:
not relevant: a prediction was performed
Preliminary study:
not relevant: a prediction was performed
Test performance:
not relevant: a prediction was performed
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Value:
0.792
Remarks on result:
readily biodegradable based on QSAR/QSPR prediction
Remarks:
(Anaerobic Linear) Biodegradation Probability - Biowin7: ≥0.5: “Fast”
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Value:
0.554
Remarks on result:
readily biodegradable based on QSAR/QSPR prediction
Remarks:
Biowin5 (MITI linear model) Biodegradation Probability
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Value:
1.035
Remarks on result:
readily biodegradable based on QSAR/QSPR prediction
Remarks:
Biowin1 (linear model) Probability of Rapid Biodegradation - Biowin1 ≥0.5: “Rapid
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Value:
0.981
Remarks on result:
readily biodegradable based on QSAR/QSPR prediction
Remarks:
Biowin2 (non-linear model) Probability of Rapid Biodegradation - Biowin2: ≥0.5: “Rapid"
Details on results:
A prediction of ready biodegradability of the substance Pyrogallol was performed by EPI BIOWIN (Biowin1, Biowin2, Biowin3, Biowin4, Biowin5, Biowin6 and Biowin7).

 TYPE

 NUM

 Biowin1 FRAGMENT DESCRIPTION  

COEFF

 VALUE

 Frag

 3

 Aromatic alcohol  [-OH]  

 0.1158

 0.3474

 MolWt

 *

 Molecular Weight Parameter

 

 -0.0600

 Const

 *

 Equation Constant  

 

 0.7475

 RESULT

 

 Biowin1 (Linear Biodeg Probability)

 

 1.0349

 TYPE

 NUM

Biowin2 FRAGMENT DESCRIPTION

 COEF

 VALUE

 Frag

 3

Aromatic alcohol  [-OH]  

0.9086

  2.7258

 MolWt

 *

Molecular Weight Parameter

 

-1.7908

 RESULT

 

Biowin2 (Non-Linear Biodeg Probability)

 

0.9810

A Probability Greater Than or Equal to 0.5 indicates --> Biodegrades Fast

A Probability Less Than 0.5 indicates --> Does NOT Biodegrade Fast

 TYPE  NUM

Biowin3 FRAGMENT DESCRIPTION

 COEF  VALUE
 Frag  3

Aromatic alcohol  [-OH]  

  0.0564

 0.1691

 MolWt

 *

Molecular Weight Parameter

 

-0.2787

 Const

 *

Equation Constant  

 

3.1992

RESULT

 

 Biowin3 (Survey Model - Ultimate Biodeg)

 

3.0896

 TYPE

 NUM

Biowin4 FRAGMENT DESCRIPTION

 COEFF

 VALUE

Frag

 3

Aromatic alcohol  [-OH]  

0.0397

0.1191

MolWt

Molecular Weight Parameter

 

-0.1820

Const

 *

Equation Constant

 

3.8477

RESULT

 

Biowin4 (Survey Model - Primary Biodeg)

 

3.7849

Result Classification:   5.00 -> hours     4.00 -> days    3.00 -> weeks

 (Primary & Ultimate)    2.00 -> months    1.00 -> longer

 TYPE  NUM

Biowin5 FRAGMENT DESCRIPTION  

 COEF

 VALUE

 Frag

 3

Aromatic alcohol  [-OH]  

 0.0642

 0.1927

 Frag

 3

Aromatic-H  

0.0082

0.0247

MolWt

 *

Molecular Weight Parameter  

 

-0.3752

Const

 *

Equation Constant

 

0.7121

RESULT

 

Biowin5 (MITI Linear Biodeg Probability)

 

 0.5543

 TYPE  NUM

Biowin6 FRAGMENT DESCRIPTION

COEF 

 VALUE

 Frag

 3

Aromatic alcohol  [-OH]

0.4884

 1.4653

 Frag

 3

Aromatic-H

0.1201

0.3604

 MolWt

 *

Molecular Weight Parameter

 

 -3.6407

RESULT

 

Biowin6 (MITI Non-Linear Biodeg Probability)

 

 0.6705

A Probability Greater Than or Equal to 0.5 indicates --> Readily Degradable

A Probability Less Than 0.5 indicates --> NOT Readily Degradable

TYPE 

 NUM

 Biowin7 FRAGMENT DESCRIPTION  

 COEF

 VALUE

 Frag

 3

Aromatic alcohol  [-OH]  

0.0807

 0.2422

 Frag

 3

Aromatic-H

-0.0954

 -0.2863

Const

 *

Equation Constant  

 

 0.8361

RESULT

 

Biowin7 (Anaerobic Linear Biodeg Prob)

 

 0.7920

A Probability Greater Than or Equal to 0.5 indicates --> Biodegrades Fast

A Probability Less Than 0.5 indicates --> Does NOT Biodegrade Fast

Ready Biodegradability Prediction: (YES or NO)

----------------------------------------------

Criteria for the YES or NO prediction:  If the Biowin3 (ultimate surveymodel) result is "weeks" or faster (i.e. "days", "days to weeks", or "weeks" AND the Biowin5 (MITI linear model) probability is >= 0.5, then the prediction is YES (readily biodegradable).  If this condition is not satisfied, the prediction is NO (not readily biodegradable).  This method

is based on application of Bayesian analysis to ready biodegradation data (see Help).  Biowin5 and 6 also predict ready biodegradability, but for degradation in the OECD301C test only; using data from the Chemicals Evaluation and Research Institute Japan (CERIJ) database.

Validity criteria fulfilled:
not applicable
Interpretation of results:
readily biodegradable
Conclusions:
The ready biodegradation property of Pyrogallol was investigated using QSAR approach with Biowin v4.10 plug-in from EPISUITE v4.1 from US EPA.
It is expected that the substance is readily biodegradable.
Executive summary:

The ready biodegradation property of Pyrogallol was investigated using QSAR approach with Biowin v4.10 plug-in from EPISUITE v4.1 from US EPA.

It is expected that the substance is readily biodegradable.

Adequacy of the QSAR:

1) QSAR model is scientifically valid.

2) The substance falls within the applicability domain of the QSAR model.

3) The prediction is fit for regulatory purpose.

The prediction is adequate for the Classification and Labelling or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006: Annex XI Section 1.3

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: To explain the darkening effect of phenol waste-water during biodegradation and to find out a suitable treatment methods
- Short description of test conditions: The investigations were performed on pyrogallol and other 12 types of phenols predominant in phenolic
waste-water; Phenol adapted activated sludge culture was seeded with raw and digested sludge taken from municipal waste treatment plants. Crude sludge was taken from primary clarifiers, while the digested sludge carne from the selected anaerobic digestion chambers.
The efficiencies of the separate technoiogicaI processes were characterized by determining phenol concentrations, COD and BOD, pH, Mohlman's index, total solid and colour of waste-water. The determinations have been performed by standard methods, including:
1. Concentrations of phenols by spectrophotometric methods [Swietloslawska et al. 1962] (spectrophotometer UNICAM, model SP850B);
2. COD, after Eckenfelder [Malina et al. 1967];
3. BOD by dilution method [Lurie et al. 1955];
4. Colour by determining the colour threshold number [Malina et al. 1967];
- Parameters analysed / observed:
1. An extent of biodegradation of the substance (which is a phenol);
2. The changes occuring in aquaeus phenol solution aerated with atmospheric oxygen in absence of microorganisms;
3. Susceptibility to biodegradation of phenol chemical oxidation products;
4. Chemical or biochemical oxidation process responsib!e for the darkening ofbiologicalIy treated phenol water.
The purpose of the paper was to solve the above problems by performing a series of experirnents.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Remarks:
Phenol adapted activated sludge culture
Details on inoculum:
Phenol adapted activated sludge culture was seeded with raw and digested sludge taken from municipal waste treatment plants. Crude sludge was taken from primary clarifiers, while the digested sludge carne from the selected anaerobic digestion chambers.
In order to obtain mixed population of microorganism, the crude and digested
sludge have been converted into activated sludg' e by applying a dircct conversion method, biomass growth and acclimation (Chmielowski et al. 1970 and 1972). To complete the nourishing composition tri-basic ammonium sulphate was added to the feed, continuously introduced into the aeration tank
Duration of test (contact time):
>= 15 - <= 120 min
Initial conc.:
200 other: mg/m3
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: COD mgO2/dm3
Parameter followed for biodegradation estimation:
other: Colour threshold number
Parameter followed for biodegradation estimation:
other: Hydraulic loading m3/m3d
Parameter followed for biodegradation estimation:
other: Sludge content g/m3
Parameter followed for biodegradation estimation:
other: Sludge loading
Remarks:
g COD/g
Parameter followed for biodegradation estimation:
other: Sludge loading
Remarks:
g BOD5/g
Parameter followed for biodegradation estimation:
other: Mohlman's index cm3/g
Parameter followed for biodegradation estimation:
other: Concentrations of Pyrogallol by spectrophotometric methods
Details on study design:
not specified
Reference substance:
not required
Parameter:
other: %degradation
Value:
64
Remarks on result:
other: % observed after 63 days
Remarks:
pyrogallic acid degrades in activated sludge
Parameter:
other: Colour threshold number
Remarks on result:
other: the coloured products of degradation appeared
Parameter:
COD
Value:
0.1 other: g COD/g
Remarks on result:
other: Sludge loading
Parameter:
BOD5
Value:
0.06 other: g BOD5/g
Remarks on result:
other: Sludge loading
Results with reference substance:
not relevant

- pH: a slight variations in pH values occuring in the course of aeration o activated sludge have been observed;

- parent substance concentration: a rapid decrease in parent substance concentration was not accompanied by a reduction in COD value;

- coulored products of degradation: the coloured products of degradation appeared: the occurence was reported below

 Time (h)  1  3
 2  40  500  500
 3  80  500  500
 4  100  800  500
 5  100  1000  500

A chemical oxidation of pyrogallol results also in formation of coloured degradation products. It may be assumed that under such conditions no

biochemical processes occur practically. This can be explained e.g. by toxic phenol concentrations for not acclìmated microorganisms which could enter the tanks during aeration process. In chemical oxidation the threshold colour number was severa! times higher than in case of biodegradation. A slight reduction in COD values, observed during chemical oxidation shows, however, that the colour intensity is associated with a light alterations in the structure of polyphenols, and a complete decay of their parent forms. The observations have confirmed the hypothesis that the colour is chiefly caused by chemical oxidation with atmospherìc oxygen, occuring in the course of activated sludge aeration.

A high susceptibility to chemical oxidation has been stated; it is, however, characteristic that a considerable amount of organic compounds, which ought to be biodegraded during aeration with the activated sludge, remained in the mixed liquor. It seems that its resistance to biodegradation by acclimated activated sludge (which probably produced the enzymes controlling the definite way of decomposition) was due to specific changes in its molecular structure caused by chemical oxidation. A considerable stabilìty of the colour resulting from chemical oxidation should be emphasized.

The performed observations have confirmed the hypothesis, that the unsatisfactory quality of biologically treated phenolic waste-water is chiefly due to chemical oxidation processes. The presence of colour products in the effiuent as well as the necessity of their eventual removal should be always taken into consideration even where the minimum amounts of oxygen are allowed in the phenolic waste-water

prior to its biodegradation.

Validity criteria fulfilled:
not specified
Interpretation of results:
inherently biodegradable
Conclusions:
Phenol adapted activated sludge was used as inoculum. The test substance was rapidly disappeared in 0.5 hour. The %COD only reached 70% after 3-6 hours. The oxidation products of the substance were reported to be persistent.
Executive summary:

The investigations have been carried out to detect the reason of poor quality of the phenolic waste-water after its biological treatment. A much advanced degradation of the mono- and polyhydroxy phenols tested has been achieved by applying acclimated activated sludge. Although in alJ secondary offluents low COD values and no phenolic substrates were stated, however coloured biodegradation products occured in case

of four polyphenols. Next, it has been shown that monohydroxy compounds and rezorcine are resistant to chemical oxidation with atmospheric oxygen during 5 hour aeration. Partial chemical oxidation yielding coloured compounds has been stated in case of hydroquinone, pyrocatechol, pyrogallol and phloroglucinol. Chemical oxidation products pyrogallol were biodegradation resistant.

Hence, chemical oxidation affects molecular structure of some phenols resulting in their biodegradability decreate. The investigations have confirmed the hypothesis that chemical oxidation processes are chiefly responsible far the unsatisfactory quality of the secondary effluent. For this the oxidation of phenolic waste-water during its transport and cooling prior to biodegradation should be avoided.

Endpoint:
biodegradation in water: screening test, other
Remarks:
Aerobic biodegradation study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: Three acclimated sludge were prepared and each acclimated sludge was tested as to its ability to metabolize pyrogallol. The purpose of these runs was to determine if there were any apparent metabolic differences among the three systems.
- Short description of test conditions:
- Parameters analysed / observed:
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: not specified
- Expiration date of the lot/batch: not specified
- Purity test date: not specified

RADIOLABELLING INFORMATION (if applicable) not applicable
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not specified
- Preliminary purification step (if any): not specified
- Final dilution of a dissolved solid, stock liquid or gel: not specified
- Final preparation of a solid: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material) solid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) not applicable

OTHER SPECIFICS: not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: acclimated activated sludges
Details on inoculum:
- Source of inoculum/activated sludge procedure: Initially, the activated sludge units were started on a synthetic sewage of the following composition : 500 p.p.m. glucose 500 p.p.m. nutrient broth 1,000 p.p.m. potasium phosphate, dibasic 325 p.p.m. ammonium phosphate 50 p.p.m. sodium chloride 50 p.p.m. calcium chloride 50 p.p.m. magnesium sulf. The synthetic sewage was seeded with 100 ml. of settled sewage as the source of micro-organisms. After a satisfac tory quantity of activated sludge had been built up and a clear supernatant produced, the systems were acclimated.
- Laboratory culture: Pseudomonas fluorescens
- Method of cultivation: not speciefied
- Storage conditions: not speciefied
- Storage length: not speciefied
- Preparation of inoculum for exposure: Three activated sludges were ac climated to a daily feed of 500 p.p.m.

- Pretreatment: not speciefied
- Concentration of sludge: The mixed liquor suspended solids were 5230 p.p.m. for the phenol unit, 3310 p.p.m. for the benzoic acid unit, unit, and 6,640 p.p.m. for the catechol unit.
- Initial cell/biomass concentration: not speciefied
- Water filtered: not speciefied
- Type and size of filter used, if any: not speciefied

Air was supplied through carborundum diffusers at a rate designed to keep the sludge in suspension
Duration of test (contact time):
12 h
Initial conc.:
500 other: ppm
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: % Theoretical Oxidation
Remarks:
by acclimated activated sludges
Details on study design:
TEST CONDITIONS
- Composition of medium:
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature: not speciefied
- pH: not speciefied
- pH adjusted: not speciefied
- CEC (meq/100 g): not speciefied
- Aeration of dilution water: not speciefied
- Suspended solids concentration: 500 ppm
- Continuous darkness: not speciefied
- Other: not speciefied

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: air was supplied through carborundum diffusers at a rate designed to keep the sludge in suspension. This quantity of air more than satisfied the biologi cal demand for oxygen
- Method used to create anaerobic conditions: not relevant: it was and aerobic biodegradation study
- Measuring equipment: not speciefied
- Test performed in closed vessels due to significant volatility of test substance: not speciefied
- Test performed in open system: Y
- Details of trap for CO2 and volatile organics if used: not speciefied
- Other: not speciefied

SAMPLING
- Sampling frequency: at the end of the study
- Sampling method: not speciefied
- Sterility check if applicable: not speciefied
- Sample storage before analysis: not speciefied
- Other: not speciefied

CONTROL AND BLANK SYSTEM
- Inoculum blank: not speciefied
- Abiotic sterile control: not speciefied
- Toxicity control: not speciefied
- Other: not speciefied

STATISTICAL METHODS: not speciefied
Reference substance:
not required
Preliminary study:
not specified
Test performance:
not specified
Parameter:
other: % Theoretical Oxidation
Value:
24
Sampling time:
12 h
Parameter:
other: % Theoretical Oxidation
Value:
16
Sampling time:
12 h
Parameter:
other: % Theoretical Oxidation
Value:
23
Sampling time:
12 h
Validity criteria fulfilled:
not specified
Interpretation of results:
other: aerobic biodegradation observed
Conclusions:
In an aerobic biodegradation study, pyrogallol degrades in activated sludge at varying rates, such as 16 to 24% after a half day in high inoculum concentrations.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: The tests and methods for the determination of biological degradability have been discussed by many authors (Fischer, 1973; Ludzack and Ettinger, 1963; Pitter, 1971).
- Short description of test conditions: The test was performed in a batch system. The tested substance is dissolved in a beaker in a biological medium in a concentration corresponding to 200mg/L (initial concentration) COD (based on). The tested substance is a sole source of organic carbon for the microbes of the inoculum. Oxygen conditions: aerobic. Inoculum or test system: activated sludge, adapted. Details on inoculum: Activated sludge taken from a sewage plant was cultivated for the test.
- Parameters analysed / observed: biological degradability of organic substances
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
- Laboratory culture:
- Method of cultivation:
- Storage conditions:
- Storage length:
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge:
- Initial cell/biomass concentration:
- Water filtered: yes/no
- Type and size of filter used, if any:
Initial conc.:
200 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium:
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:
Reference substance:
not required
Parameter:
other: Rate of biodegradation
Remarks:
mg COD g-1 h-1
Value:
0
Remarks on result:
not determinable
Details on results:
% Degradation

Parameter: % degradation (DOC removal)
Value:
Sampling time: 5 d

Details on results: The rate of biodegradation based on COD removal was ... mg COD/gram per hour.
Parameter:
COD
Value:
40 other: %
Remarks on result:
other: Percent removed
Remarks:
(based upon COD)

On the basis of the knowledge obtained, the factors affecting biological degradability can be divided into three groups:

1. physico-chemical factors (temperature, solubility, degree of dispersion of the compound in the medium, pH, dissolved oxygen),

2. biological factors (history of the microbial culture, its age, manner and time of its adaptation, toxicity of the compound, effect of other substrates) and

3. chemical factors (size of molecule, length of chain, kind, number and position of substituents in the molecule, stereochemistry).

Validity criteria fulfilled:
not specified
Interpretation of results:
not readily biodegradable
Conclusions:
pyrogallol is a substance biologically hard to decompose.
Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were a sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. The rate of degradation was expressed in terms of mg of COD removed by a g of the initial dry matter of inoculum h- t.

Endpoint:
biodegradation in water: screening test, other
Remarks:
Anaerobic Biodegradation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
- Principle of test:
- Short description of test conditions:
- Parameters analysed / observed:
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Sludge samples were collected from primary or secondary anaerobic digesters in 1- or 2-liter jars, tightly capped
- Laboratory culture: not specified
- Method of cultivation: Sludges were from waste treatment plants in the following mid-Michigan communities: Adrian, Ann Arbor, Chelsea, Holt, Ionia, Jackson, Mason, Portland, and St. Johns.
- Storage conditions: stored at 4°C until use.
- Storage length: not specified
- Preparation of inoculum for exposure: not specified
- Pretreatment: not specified
- Concentration of sludge: Percent organic matter (total solids x volatile solids) varied from 0.89% (Holt) to 1.99% (Jackson) with a median value of 1.53%. Average retention times varied from 17 (Ionia) to 39 (Holt) days with a median value of 20 days.
- Initial cell/biomass concentration: not specified
- Water filtered: yes/no not specified
- Type and size of filter used, if any: not specified

Inflow varied from 1.6 x 106 (Chelsea) to 68 x 106 (Jackson) liters/day with a median value of 4.4 x 106 liters/day
Details on study design:
TEST CONDITIONS
- Composition of medium:
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:
Preliminary study:
Preliminary experiments were initiated to obtain degradation data for a wide variety of organic compounds to select compounds for future testing of assay parameters and to determine the minimum length of incubation.
Test performance:
Compounds were initially incubated for 4 weeks; however, this proved to be inadequate. Subsequently, all compounds were incubated for 8 weeks, or until net methane production had ceased.
Key result
Parameter:
other: %degradation
Value:
> 75
Sampling time:
56 d
Details on results:
Of 94 compounds tested, Pyrogallol was mineralized (>75% of theoretical methane production) in at least one sludge
Validity criteria fulfilled:
yes
Conclusions:
Pyrogallol degrades at varyious rates under anaerobic conditions with >75% after 56 days in sludge.
Executive summary:

A simple, generalized method was refined and validated to test whether an organic chemical was susceptible to anaerobic degradation to CH4 + CO2. The method used digested sewage sludge diluted to 10% and incubated anaerobically in 160-ml serum bottles with 50 µg of C per ml of test chemical.

Biodegradation was determined by the net increase in gas pressure in bottles with test chemicals over the pressure in nonamended sludge bottles. Gas production was measured by gas chromatography and by a pressure transducer. The latter method is recommended because of its speed, accuracy, and low cost.

Sewage sludge from municipal digesters with 15- to 30-day retention times was found to be suitable. The sludge could be stored anaerobically at 4°C for up to 4 weeks with satisfactory test results.

Description of key information

Key value for chemical safety assessment

Additional information