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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 April - 11 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 20422
- Substance type: Pure active substance
- Physical state: Volatile liquid (VP 87.5 kPa at 20 °C)
- Analytical purity: 99.99%
- Expiration date of the lot/batch: 16 Feb 2013
- Stability under test conditions: Reactive to moisture
- Storage condition of test material: Sealed container

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Ssecondary effluent freshly obtained from a municipal sewage treatment plant:
'Waterschap Aa en Maas','s-Hertogenbosch, The Netherlands
- Preparation of inoculum for exposure: Secondary effluent was filtered through a coarse filter paper, the first 200 ml was discarded. The filtrate was kept aerated until inoculation.
- Inoculation level: Four mL/L, ca 2.4 mL in test bottles
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
ca. 50 - ca. 80 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium, concentrations as per OECD 301D
- Solubilising agent (type and concentration if used): None
- Test temperature: 21.8 °C to 22.4 °C.
- pH: 7.4 - 7.5 at test initiation
- pH adjusted: No
- Aeration of dilution water: Dilution water not used, mineral medium was left at room temperature overnight before test, DO concentration measured before test began to assure saturation.
- Continuous darkness: yes
- Other: Filtered sludge aerated until inoculation, ca. 2.4 mL aerated sludge added per test bottle.

TEST SYSTEM
- Culturing apparatus: ca. 300 mL BOD bottles with glass stoppers
- Number of culture flasks/concentration: Two per concentration per day.
- Method used to create aerobic conditions: Aeration of medium before inoculation
- Measuring equipment: WTW inolab Oxi 730 supplied with a WTW CellOx 325
oxygen electrode, electrolyte type ELY/G.
- Test performed in closed vessels due to significant volatility of test substance: yes

SAMPLING
- Sampling frequency: At day 0, 7, 14, 21 and 28.
- Sampling method: Stopper removed, oxygen electrode inserted and dissolved oxygen concentration measured. pH in sample botles measured at start of test only.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (ten bottles)
- Abiotic sterile control: No
- Toxicity control: Yes (six bottles), same amount of test and reference substances combined as in test substance low concentration (50 mg/L) and reference substance (2 mg/L) bottles separately.

STATISTICAL METHODS:
ThOD and BOD calculated per OECD guidelines
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (O2 consumption)
Value:
ca. 0
Sampling time:
28 d

BOD5 / COD results

Results with reference substance:
79% biodegradation of reference substance on day 7.
Toxicity was assessed by comparison of toxicity test bottle BOD with sum of reference substance and low test substance BOD results. No difference was observed (see Table 1), so the test substance was not considered to inhibit activated sludge.

Any other information on results incl. tables

Table 1, pH at the start and oxygen concentration during the test

Series

Content

pH at τ=0

Flask No.

Oxygen concentration (mg O2/L) after x days

0

7

14

21

28

Inoculum blank

Mineral medium,

7.5

A

8.52

8.68

8.11

7.83

7.63

inoculum

B

8.54

8.66

8.17

7.79

7.64

mean

8.53

8.67

8.14

7.81

7.64

Procedure control

Mineral medium,

7.5

A

8.57

7.47

7.00

inoculum

B

8.56

7.48

7.10

sodium acetate (2 mg/l)

mean

8.57

Test substance, low

Mineral medium,

7.5

A

8.57

8.69

8.10

7.94

7.68

inoculum

B

8.58

8.77

8.24

7.80

7.73

CAS# 756-12-7

mean

8.58

= 10 μl/BOD bottle

= 15.6 mg/BOD bottle

= approx. 50 mg/l

Test substance, high

Mineral medium,

7.4

A

8.60

8.68

8.05

7.92

7.64

inoculum

B

8.59

8.71

8.14

7.90

7.67

CAS# 756-12-7

mean

8.60

= 15 μl/BOD bottle

= 23.4 mg/BOD bottle

= approx. 80 mg/l

Toxicity control

Mineral medium,

7.4

A

8.55

7.41

6.97

inoculum

B

8.56

7.44

6.97

CAS# 756-12-7

mean

8.56

= approx. 50 mg/l plus

sodium acetate

(2 mg/l)

 

Table 2, Mean values of oxygen depletion at different points in time ¹

Series

Content

Flask No.

Oxygen depletion (mg BOD/L) after x days

7

14

21

28

Procedure control

Mineral medium,

A

1.24

1.18

inoculum

B

1.23

1.08

sodium acetate (2 mg/l)

Test substance, low

Mineral medium,

A

0.03

0.09

-0.09

0.00

inoculum

B

-0.05

-0.05

0.06

-0.05

CAS# 756-12-7

= 10 μl/BOD bottle

= 15.6 mg/BOD bottle

= approx. 50 mg/l

Test substance, high

Mineral medium,

A

0.05

0.15

-0.04

0.06

inoculum

B

0.02

0.06

-0.03

0.03

CAS# 756-12-7

= 15 μl/BOD bottle

= 23.4 mg/BOD bottle

= approx. 80 mg/l

Toxicity control

Mineral medium,

A

1.29

1.20

inoculum

B

1.26

1.20

CAS# 756-12-7

= approx. 50 mg/l plus

sodium acetate

(2 mg/l)

1, Values corrected for oxygen depletion in blank control (mean value) 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
O2 depletion in the inoculum blank was below 1.5 mg O2/l after 28 days, residual O2 in test bottles > 0.5 mg/l at any time, control substance degraded >60% within 14 days, differences of duplicate biodegradation values less than 20% as mg O2/L
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
CAS# 756-12-7 is not readily biodegradable
Executive summary:

Biodegradation of CAS# 756-12-7 was assessed in a closed bottle test (OECD TG301D) conducted under GLP criterion. Sodium acetate was used as reference. CAS# 756-12-7 did not measurably degrade during the test period. In toxicity controls, CAS# 756-12-7 at a concentration of 50 mg/L did not measurably prevent the reference substance from being degraded. CAS# 756-12-7 is not readily biodegradable.

The study was conducted in accord with internationally accepted guidelines under GLP criteria. It is reliable without restriction and is suitable for use in Risk Assessment, Classification & Labelling, and PBT Analysis