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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:
16-09-2015 to 15-10-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (10-09-2015). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
DEIONISED WATER
Deionized water containing no more than 0.01 mg/L Cu (ISO 17025 certified; non-GLP analysis) was prepared in a water purification system.

TEST BOTTLES
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

NUTRIENTS, STOCKS AND ADMINISTRATION
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was not added to the river water to prevent nitrification. Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top with aluminum foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.

TEST PROCEDURES
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and silica gel with test substance, 6 bottles with river water and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

TEST CONDITIONS
The pH of the media was 8.0 at the start of the test. The pH of the medium at day 28 was 7.8 (test) and 8.0 (controls). Temperatures were within the prescribed temperature range of 22 to 24°C.

CALCULATION OF BIODEGRADATION PERCENTAGES
The biodegradation was calculated as the ratio of the BOD to the theoretical oxygen demand (ThOD). The calculated theoretical oxygen demand (ThOD) of the test substance (mono-constituent) is 2.7 mg/mg. The ThOD of the reference substance sodium acetate is 0.8 mg/mg.
Reference substance:
acetic acid, sodium salt
Test performance:
VALIDITY OF THE TEST
The validity of the test is demonstrated by an endogenous respiration of 1.4 mg/L at day 28 (Table I). Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 89. Finally, the validity of the test is shown with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

TOXICITY
Inhibition of the endogenous respiration of the inoculum by the test substance tested was not detected. Therefore, no inhibition of the biodegradation due to the initial concentration of the test compound is expected.
Key result
Parameter:
% degradation (O2 consumption)
Value:
80
Sampling time:
28 d
Details on results:
Over 60% biodegradation was achieved in a period of 10 days immediately following the attainment of 10% biodegradation. The test item therefore fulfilled the time window criterion for ready biodegradable compounds. Hence the test substance should be classified as readily biodegradable.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 89.

Tabel 1: Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)

Oxygen concentration (mg/L)

Ocs

Ot

Oc

Oa

0

8.7

8.7

8.7

8.7

 

8.7

8.7

8.7

8.7

Mean

8.7

8.7

8.7

8.7

7

8.0

6.7

7.9

3.5

 

7.9

6.8

8.0

3.9

Mean

8.0

6.8

8.0

3.7

14

7.7

4.3

7.6

2.7

 

7.7

3.8

7.6

2.9

Mean

7.7

4.1

7.6

2.8

21

7.4

3.4

7.4

-

 

7.4

3.3

7.6

-

Mean

7.4

3.4

7.5

-

28

7.3

2.8

7.3

-

 

7.2

3.2

7.3

-

Mean

7.3

3.0

7.3

-

Ocs: River water with nutrients and silica gel.

Ot: River water with nutrients, test material (2.0 mg/L) and silica gel.

Oc: River water with nutrients.

Oa: River water with nutrients and sodium acetate (6.7 mg/L).

Table 2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance and the reference substance

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

0

0.0

0.0

0

0

7

1.2

4.3

22

80

14

3.6

4.8

67

89

21

4.0

-

74

-

28

4.3

-

80

-

Validity criteria fulfilled:
yes
Remarks:
see 'Test performance'.
Interpretation of results:
readily biodegradable
Conclusions:
The substance showed 80% biodegradation in an OECD TG 301D test. The 10-day window criterion was met.
Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. The test substance did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. Veilex 1 (monoconstituent) was biodegraded by 80% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of 10 days immediately following the attainment of 10% biodegradation. Hence, this substance should be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 89% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Veilex 1
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Information based on read across
Justification for type of information:
See attached Justification and Endpoint summary.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read across information is provided
GLP compliance:
no
Specific details on test material used for the study:
As provided in the read across document
Inoculum or test system:
other: See source substance
Key result
Parameter:
% degradation (O2 consumption)
Value:
80
Remarks on result:
other: Based on read across information
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Veilex 2 is readily biodegradable based on the information of Veilex 1
Executive summary:

For Veilex 2 no ready biodegradability information is available. For Veilex 1 a well conducted ready biodegradation test is available showing ready biodegradation. In view of the similarities between these substances Veilex 2 can be considered readily biodegradable.

Description of key information

Veilex 2 is readily biodegradable based on biodegradability information from the structural analogue Veilex 1 (CAS# 63449-88-7).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Veilex 2 is considered readily biodegradable based on information from the analogue Veilex 1. The study with Veilex 1 is presented first below and thereafter the read across justification is presented.

The ready biodegradability of Veilex 1 was investigated in a study conducted in accordance with OECD TG 301D (Closed Bottle Test) and GLP. The concentration tested was 2.0 mg/l test substance. The inoculum used was river water which was aerated for 7 days before use to reduce the endogenous respiration. In addition, particles were removed by sedimentation after 1 day while moderately aerating. Under these conditions the test substance biodegrades 80% in 28 days. The 10-day window criterion for ready biodegradability was met. Inhibition of the endogenous respiration of the inoculum by the test substance was not detected. The validity of the test is demonstrated by an endogenous respiration of 1.4 mg/L at day 28 . Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 89. Finally, the validity of the test is shown with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period. Veilex 2 is considered to have similar potential for biodegradation. Therefore Veilex 2 is assessed as readily biodegradable.

Biodegradation of Veilex #2 (CAS #13487-27-9) based on read across from data available for Veilex #1 (CAS #63449-88-7).

Introduction and hypothesis for the analogue approach

Veilex#1 has a cyclohexylethyl backbone with a butyrate ester group attached. For this substance there are no experimental biodegradation data available. Biodegradation information may be generated by other means, i.e. applying alternative methods such as QSARs, grouping and read-across. For assessing the ready biodegradability of Veilex#2 the analogue approach is selected because for a closely related analogue, Veilex #1 ready biodegradation information is available which can be used for read across.

Hypothesis: Veilex#2 has the same biodegradation potential as Veilex #1, because structurally, the only difference between Veilex #2 and Veilex #1 is the length of the alpha-alkyl chain connected to the ester moiety.

Available information: No experimental biodegradation data are available for the target chemical Veilex #2. For Veilex #1 a ready biodegradation study was performed according to OECD TG 301D and in compliance with GLP criteria (IFF, 2015). In this study Veilex #1 was biodegraded by 80% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of 10 days immediately following the attainment of 10% biodegradation. Hence, this substance is classified as readily biodegradable. The data are reliable without restrictions (Klimisch 1).

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical are shown in the data matrix, including physico-chemical properties and available ecotoxicological information.

Purity / Impurities

Both Veilex #2 and Veilex #1 are mono-constituent substances with purities close to 100%. Both substances do not contain any impurities that will affect the read across for biodegradation.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group.

In accordance with ECHA (2015, RAAF) Veilex #1 was selected as an analogue because of the close structural relation with Veilex #2. Other cyclohexyl-esters can be readily biodegradable too e.g. Terpinyl acetate but it depends on the presence of e.g. quaternary C’s, which may limit the degradation or in case the ester is a formate and formic acid is formed after degradation (e.g. CP Formate.

Structural similarities and differences: Veilex #2 and Veilex #1 have the same backbone and the same functional group. In Veilex #2 an acetic-ester group is attached to the ethyl moiety, while at Veilex #1 this is a butyric-ester group. This two carbon difference will not make a difference in the ready biodegradability of both substances, because acetic and butyric acid are both readily biodegradable. This is supported with the BIOWIN predictions presented below.

BIOWIN predictions: BIOWIN (v4.10) predictions are made for both Veilex #2 and Veilex #1 to provide further insight on the biodegradation potential of these substances and the possible effect of structural differences on biodegradation. Only the BIOWIN modules 5 (‘MITI-linear’) and 6 (‘MITI-non-linear’) are used because these two models are based on ready biodegradability tests, which is the endpoint to be predicted.

Table 1 BIOWIN predictions for Veilex #2 and Veilex #1 and the contribution of the fragments for the prediction of ready and non-ready biodegradability

 

 

Veilex 2

 

Veilex 1

 

Fragment

BIOWIN 5

BIOWIN 6

BIOWIN 5

BIOWIN 6

Ester

0.3437

2.4462

0.3437

2.4462

Methyl *2

0.0008

0.0389

0.0008

0.0389

-CH2- (linear) * 2

-

-

0.0988

0.8590

-CH- (linear)

-0.0507

-0.0998

-0.0507

-0.0998

-CH2- (cyclic) *5

0.0986

1.1826

0.0986

1.1826

-CH- (cyclic)

0.0124

-0.1295

0.0124

-0.1295

MW parameter

-0.5065

-4.9150

-0.5900

-5.7249

Equation constant

0.7121

-

0.7121

-

RESULT

0.6106

0.7406

0.6260

0.7499

From these BIOWIN predictions it can be seen that both Veilex #2 and Veilex #1 are readily biodegradable with values of well above the cut-off value of 0.5.

Uncertainty of the prediction: There are no remaining uncertainties, the additional two C-atoms in the alkyl chain are not affecting the ready biodegradability potential as is further supported with the BIOWIN predictions. In view of the minimal differences in structures a score 5 can be assigned when using the criteria according to RAAF (ECHA, 2015, RAAF).

Conclusions for environmental fate and classification and labelling

Environmental fate: Veilex #1 is readily biodegradable and therefore Veilex #2 will be assessed as readily biodegradable as well.

Classification and Labelling: This ready biodegradation results in the assignment of ‘rapidly degradable’ for purpose of classification and labelling. 

Data matrix for read across to Veilex #2 and from Veilex #1

Common name

Veilex #2

Veilex #1

Chemical name

α-methylcyclohexylmethyl acetate

1-cyclohexylethyl butyrate

Chemical structures

CAS no

13487-27-9

63449-88-7

Empirical formula

C10H18O2

C12H22O2

Physico-chemical data

 

 

Molecular weight

170

198

Physical state

liquid

liquid

Melting point,oC

<-20

< -20

Boiling point,oC

215.7

246.5

Vapour pressure, Pa

38.5 (at 24°C)

6.5 (at 24 °C)

Water solubility, mg/L

43.0 (at 24°C)

23.8 (at 24 °C)

Log Kow

3.2

5.2

Fate and behaviour

 

 

Biodegradation

Read across from Veilex #1:

Readily biodegradable

Used for read across:

Readily biodegradable OECD TG 301D