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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:
03 May 1985 to 14 June 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EPA Chemical Fate Test Guidelines, publ. No. EPA 560/6-83-003, Method CG-2000 (Aerobic Aquatic Biodegradation) 1982.
Deviations:
not specified
GLP compliance:
yes
Remarks:
Good Laboratory Practice Regulations set forth in 40 CFR Part 792.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: Batch I

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The stability of the test material with regard to aquatic biodegradation is the subject of this report.

FORM AS APPLIED IN THE TEST (if different from that of starting material): colorless liquid (not different)

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
other: mixture of natural sediment (freshwater) and activated sludge (domestic)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sediment: pond sediment was obtained as a dredge sample from Colonial Park, East Millstone, New Jersey; activated sludge: sludge was obtained fresh from the Raritan Valley-Somerset Sewage Authority, Bridgewater, New Jersey.
- Method of cultivation: Acclimation Phase (May 3, 1985 to May 17, 1985): The acclimation medium was prepared by adding the following to a 2L Erlenmeyer flask: 1 mL each of mineral salt Solutions I, II, and III, 10.0334 g of sediment, 100 mL of activated sludge, 900 mL of glass distilled water, 25.7 mg casamino acids and 25.9 mg yeast extract. The mixture was homogenized in a Waring blender for 5 minutes and then filtered through glass wool. On days 0, 7, and 11; 9, 10 and 10 µL (specific gravity = 0.87 @ 20°C) of MRD-84-521, respectively, were added to the acclimationnmedium. The acclimation medium was shaken gently on the
gyrorotatory shaker for 13 days in the dark. The Erlenmeyer flask was stoppered with gauze which was removed only at times of test material addition.
- Storage conditions: dark
- Storage length: 13 days
- Preparation of inoculum for exposure: On day 13 the contents of the acclimation medium flask were poured into an Imhoff settling cone and were allowed to settle overnight. The resulting supernatant served as the inoculum for the biodegradation phase test systems.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
>= 12.2 - <= 13 mg/L
Based on:
test mat.
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 mL each of mineral salt Solutions, 100 mL of adapted inoculum and 900 mL of aerated glass distilled water.
- Mineral Salt Solutions: I: NH4CI (35 g/L), KNO3 (15 g/L), K2HPO4 x 3H2O (76 g/L), NaH2P04 x H20 (26 g/L); II: KCl (10 g/L), MgS04 (20 g/L), FeS04 x 7H20 (1 g/L); III: CaCl2 (5 g/L), ZnCl2 (0.05 g/L), MnCl2 x 4H20 (0.5 g/L), CuCI2 x 2H2O (0.07 g/L), CoCl2 x 6H20 (0.003 g/L), H3BO3 (0.001 g /L), MoO3 (0.0004 g/L)
- Test temperature: 21.5 - 25 °C
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 L Gledhill flasks
- Number of culture flasks/concentration: 3 replicates
- Method used to create aerobic conditions: At each sampling point the flask contents were sparged with air through the sidearms for 5 minutes.
- Measuring equipment: As more carbonate is formed, the trap solution becomes more acidic so that less HCI titrant is required to reach the equivalence point.
- Test performed in closed vessels: The eight flasks were sealed.
- Details of trap for CO2 and volatile organics if used: 10 mL of 0.2N sodium hydroxide (NaOH) were added to the center reservoir of each flask.

SAMPLING
- Sampling frequency: Day 4, 7, 11, 14, 18, 21, 25 and 28.
- Sampling method: The flasks were removed from the shaker and the spent NaOH was emptied from the reservoirs, combined with 2x10 mL rinsings of distilled water into 125 mL Erlenmeyer flasks. A few drops of phenolphthalein indicator were added to the spent solutions and they were titrated against standardized 0.1 N hydrochloric acid (HCI, standardized 0.101 N HCI was also used) to a clear endpoint. The amount of HCI required to reach the endpoint was recorded. Ten mL of fresh 0.2N NaOH were added to each reservoir and the flask contents were sparged with air through the sidearms for 5 minutes. The flasks were resealed with rubber septums and they were returned to the gyrorotatory shaker and agitated in the dark.
Day 28: The flask contents were acidified to a pH of less than 3 with concentrated H2SO4 and agitated on the shaker for four more hours. Ten mL TOC sample aliquots were removed from the flask contents and analyzed for total organic carbon (TOC). The flasks were then removed from the shaker and the reservoir contents were treated as on day 4, 7, 11, ... etc.
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 replicates
- Toxicity control: Toxicity control: Positive control Phthalic acid: 103.8 mg/L and negative Control HgCl2: 51 g/L

STATISTICAL METHODS:
Reference substance
Reference substance:
other: phthalic acid

Results and discussion

Preliminary study:
no
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
31
Sampling time:
28 d
Details on results:
The CO2 evolution rate for test material mineralization was found to be 1.5 x 10^-2/day, which corresponds to a biodegradation half-life (t1/2) of 46.2 days.

BOD5 / COD results

Results with reference substance:
Loss of phthalic acid from the positive control flask: 43.4 % based on CO2 evolution; 100.7 % based on TOC removal (28 days)
The results based on CO2 evolution are lower than those based on TOC removal for the positive control. This difference is probably a result of the incorporation of carbon into biomass. This theory is substantiated by findings that mineralization is clearly favored under the conditions of a high inoculum and this procedure incorporates a fairly low inoculum.
The data for the negative control flask indicates that the presence of HgCl2 inhibited degradation.

Any other information on results incl. tables

Table 1: Amount (mg) of Carbon evolved as Carbon Dioxide (1)

Test system

Day 4

Day 7

Day 11

Day 14

Day 18

Day 21

Day 25

Day 28

Mean – Test (2)

0.12

0.52

0.72

0.41

0.61

0.20

0.25

0.14

Cumulative Test

0.12

0.64

1.36

1.77

2.38

2.58

2.83

2.97

Positive Control – Phthalic acid

1.32

0.68

0.15

 - (3)

0.15

0.04

0.01

0.04

Cumulative Positive Control

1.32

2.00

2.15

2.15

2.30

2.34

2.35

2.39

Negative Control – HgCl2 inhibited

-        

-        

-        

-        

-        

 -

 -

 -

Cumulative negative Control

-        

-        

-        

-        

-        

 -

 -

 -

Temperature °C

25.0

22.0

23.0

22.0

22.0

21.5

21.5

22.5

(1) Calculated using the mean daily control values (Appendix II)

(2) Mean from Test A, B, and C daily values

(3) CO2 evolved was less than the mean daily control values

Table 2: Total carbon, total carbon dioxide evolution, percent material degradation, and percent TOC removal

Flask Solutions

TOC day 1 (mg/L)

TOC day 28 (mg/L)

Cumulative carbon evolved (mg) as CO2 day 28 (Table 1)

Percent material degraded day 28

Percent TOC removal day 28

Positive Control – Phthalic acid

6.436

0.714

2.39

43.4 % (b)

107 %

Negative Control – HgCl2 inhibited

6.545

5.737

0.0

0.0 % (b)

11.3 %

Mean Control

0.924

0.750

 -----

 

 

 

Test substance added (mg)

Carbon added (mg) (a)

 

Mean – Test

13.0

9.6

2.97

31 % (c)

 

(a) Calculated as: Test substance added (mg) x 0.74 = carbon added (mg)

(b) Eq. 1

(c) Eq. 1a

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Interpretation of results:
not readily biodegradable
Conclusions:
These results suggest that the biodegradation of the test substance was moderate under the conditions of the test. The substabce is not considered as rapidly biodegradable according to Regulation (EC) No. 1272/2008.
Executive summary:

Based on the test results (31 % based on CO2 evolution) for ready biodegradation, the substance was determined moderately biodegradable after 28 d. The OECD criteria for ready biodegradation are not fulfilled.