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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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test procedure according to OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal activated sludge from laboratory wastewater treatment plant fed with municipal sewage and synthetic wastewater.
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
> 0 - < 10
Sampling time:
3 d
Parameter:
% degradation (O2 consumption)
Value:
> 10
Sampling time:
4 d
Parameter:
% degradation (O2 consumption)
Value:
> 60 - < 70
Sampling time:
12 d
Parameter:
% degradation (O2 consumption)
Value:
> 70 - < 80
Sampling time:
21 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
>= 70 - <= 80
Sampling time:
28 d

ThOC mg/g = 742*

COD mg/g = 2177

ThOD mg/g = 2527*

(* = at 99.7 % purity)

Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25/05/2001 - 01/08-2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study according to international guideline (ISO 11734: 1995), validity criteria met.
Qualifier:
according to
Guideline:
other: ISO 11734: 1995 Ultimate biodegradability in digested sludge
Deviations:
yes
Remarks:
the test was extended to 79 days instead of 60 days
GLP compliance:
no
Remarks:
but declared in accordance with Swiss quality system SN EN 45001
Oxygen conditions:
anaerobic
Inoculum or test system:
digested sludge
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): digested sludge from the digester of a biological waste water treatment plant (ARA Werdhölzli, Zürich, May 8th, 2001)
- Storage length: Incubation overnight
- Preparation of inoculum for exposure: Prior to the test the sludge was washed twice with de-aerated tap water and once with de-aerated mineral medium.
- Pretreatment: incubation overnight without adaptation, no pre-conditioning
- Concentration of sludge: 2 g/L (dry weight) in final mixture
Duration of test (contact time):
79 d
Initial conc.:
136.1 mg/L
Based on:
test mat.
Initial conc.:
101.4 mg/L
Based on:
other: TOC
Details on study design:
TEST CONDITIONS
- Composition of medium: Anaerobic mineral salts medium prepared with double distilled water (conductivity <1.5 µS/cm; DOC <0.3 mg/L).

Medium contains: K2HPO4 0.27 g/L; Na2HPO4.2H2O 1.12 g/L; NH4Cl 0.53 g/L; CaCl2.2H2O 0.075 g/L; MgCl2.6H2O 0.1 g/L; FeCl2.4H2O 0.02 g/L; Resazurin 0.001 g/L; Na2S.9H2O 0.1 g/L and 10 mL/L of a stock solution containing: MnCl2.4H2O 0.05 g/L; H3BO3 0.005 g/L; ZnCl2 0.005 g/L; CuCl2 0.003 g/L; Na2MoO4.2H2O 0.001 g/L; CoCl2.6H2O 0.10 g/L; NiCl2.6H2O 0.01 g/L; Na2SeO3 0.005 g/L.

- Additional substrate: No
- Solubilising agent: No
- Test temperature: 35 ± 2 °C
- pH: at the end of the test in vessels with blank control: 7.1; with reference substance : 6.8; with 2-Ethylhexyl 4-methoxycinnamate: 6.8
- Aeration of dilution water: medium was de-aerated
- Suspended solids concentration: 2g/L dry matter

TEST SYSTEM
- Culturing apparatus: Tempure controlled incubator
- Number of culture flasks/concentration: test suspension: 3; inoculum blank: 3; procedure control: 2
- Method used to create anaerobic conditions: Incubation with anaerobic sludge
- Measuring equipment: The pressure in the headspace, representing the produced inorganic carbon (CH4 and CO2) was analyzed with a pressure measuring device (MP340A, EIRELEC Ltd).
- Test performed in closed vessels due to significant volatility of test substance: yes; System was closed to retain produced gas in headspace
- Test performed in open system: No
- Other: Resazurin (oxygen indicator) present in medium to confirm absence of oxygen

SAMPLING
- Sampling frequency: weekly after 28 days, once every 1 - 5 days before 28 days
- Sampling method: Measuring pressure with equipment described above
- Sample storage before analysis: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: No
- Other: inoculum control with reference substance diethylene glycol
Reference substance:
diethylene glycol
Remarks:
at 100 mg/L (as TOC)
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
67
Sampling time:
79 d
Remarks on result:
other: total in headspace and liquid
Parameter:
% degradation (inorg. C analysis)
Value:
58
Sampling time:
60 d
Remarks on result:
other: in headspace only
Details on results:
Based on the data of a pressure-measuring device 2-Ethylhexyl 4-methoxycinnamate reached a biodegradability of 58 and 62 % after 60 and 79 days of incubation, respectively, calculated with respect to the inorganic carbon production in the headspace. The plateau was reached after about 70 days of incubation. Including the produced inorganic carbon in the liquid phase, measured at the end of the incubation period, a total biodegradability of 2-Ethylhexyl 4-methoxycinnamate of 67 % was attained within the incubation period of 79 days. An adaptation period (lag phase) of about 10 days was observed.
Results with reference substance:
The positive control, diethyleneglycol, reached a plateau with respect to the inorganic carbon production in the headspace of 64 % biodegradation after about 35 days of incubation. Including the produced inorganic carbon in the liquid phase, measured at the end of the incubation period, a total biodegradability of the reference compound of 78 % was attained. The test was considered valid, since a plateau of more than 60 % was reached within 60 days of incubation.
Validity criteria fulfilled:
yes
Remarks:
Reference substance reached > 60% biodegradation
Interpretation of results:
other: ultimately anaerobically biodegradable
Conclusions:
The anaerobic degradation of 2-Ethylhexyl 4-methoxycinnamate was investigated. A plateau phase of more than 60 % was attained and thus 2-Ethylhexyl 4-methoxycinnamate can be termed as ultimately anaerobically biodegradable. 2-Ethylhexyl 4-methoxycinnamate showed 67 % degradation after 79 days of incubation.
Executive summary:

The anaerobic degradation of 2-Ethylhexyl 4-methoxycinnamate was investigated according to ISO 11734: 1995. A plateau phase of more than 60% was attained and thus 2-Ethylhexyl 4-methoxycinnamate can be termed as ultimately anaerobically biodegradable. 2-Ethylhexyl 4-methoxycinnamate showed 67 % degradation after 79 days of incubation. Reference substance degraded 78 % after 79 days.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20/07/1994 - 18/08/1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study according to international guideline (OECD Guideline 301F) under GLP conditions, validity criteria met.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Fresh activated sludge from a waste water teatment plant treating predominantly domestic waste (city of Geneva, Aïre) was used.
- Preparation of inoculum for exposure: The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 100 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.
- Pretreatment: no pretreatment
- Concentration of sludge: 30 mg/L (dry weight)
- Water filtered: Deionised water was used.
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: Medium is made by mixing 50 mL of solution A and 2000 mL deionised water, adding 5 mL of the solutions B, C and D and making up to 5 litres with deionised water.

Solution A: KH2PO4 8.5 g/L; K2HPO4 21.75 g/L; Na2HPO4.2H2O 33.4 mg/L; NH4Cl 0.5 g/L
Solution B: CaCl2: 27.5 g/L
Solution C: MgSO4.7H2O: 22.5 g/L
Solution D: FeCl3.6H2O 0.25 g/L; concentrated HCl one drop/L

- Test temperature: 20 °C
- pH: 7.36 - 8.54, no increase greater than 1.02 units during test
- pH adjusted: yes if necessary, to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide
- Suspended solids concentration: 2.988 g/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Sapromat D12, Voith
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: inoculum was well aerated aerobic sludge
- Measuring equipment: Sapromat
- Test performed in closed vessels due to significant volatility of test substance: No. Vessels were closed to allow measurement of gas pressure.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: 2 g of soda lime is placed in an attachment of the stopper.

SAMPLING
- Sampling frequency: not applicable - continuous recording of the oxygen replenishment

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: not included
- Toxicity control: Yes
Reference substance:
aniline
Remarks:
At 100 mg/L, purity >99.5%
Parameter:
% degradation (O2 consumption)
Value:
31
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
64
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
75
Sampling time:
21 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
78
Sampling time:
28 d
Details on results:
The biodegradation at the end of the 10-day window (days 6 to 16) is 69 %. The curves obtained with aniline alone and with 2-Ethylhexyl 4-methoxycinnamate + aniline show no toxic effect of 2-Ethylhexyl 4-methoxycinnamate on the microorganisms at the test concentration.
Blank inoculum respiration was 24 mg O/l on days 6 and 33 on day 28.
Results with reference substance:
Degradation of aniline exceeds 40 % after 7 days and 65 % after 14 days, meeting the 10-days window.
Validity criteria fulfilled:
yes
Remarks:
oxygen uptake in inoculum blank is < 60 mg/L; difference between replicates is < 20 %, aniline reference acc. to requirements
Interpretation of results:
readily biodegradable
Conclusions:
Biodegradability of 2-Ethylhexyl 4-methoxycinnamate was investigated according to OECD Guideline 301F under GLP. The biodegradation is 78 % after 28 days. The biodegradation at the end of the 10-day window (day 6 to 16) is 69 %. Thus, 2-Ethylhexyl 4-methoxycinnamate can be regarded as readily biodegradable.
Executive summary:

Biodegradability of 2-Ethylhexyl 4-methoxycinnamate was investigated according to OECD Guideline 301F under GLP. The biodegradation is 78 % after 28 days. The biodegradation at the end of the 10-d window (day 6 to 16) is 69 %. Thus, 2-Ethylhexyl 4-methoxycinnamate can be regarded as readily biodegradable. Degradation of the reference substance aniline exceeds 40 % after 7 days and 65 % after 14 days. The curves obtained with aniline alone and with 2-Ethylhexyl 4-methoxycinnamate + aniline show no toxic effect of 2-Ethylhexyl 4-methoxycinnamate on the microorganisms at the test concentration.

Description of key information

Readily biodegradable: 78 % (BOD) in 28 days, 10-day window met (OECD TG 301F)

Ultimately anaerobically biodegradable: plateau at 67 % after 79 days (ISO 11734: 1995)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Ethylhexyl Methoxycinnamate was found to be readily biodegradable in two separate studies. The results of the studies were 78 % biodegradation in 28 days (OECD TG 301F) and 70 - 80 % biodegradation in 28 days (OECD TG 301F). The poor water solubility does not seem to limit the rate of degradation to a significant extent. The key study was chosen based on the highest reliability score. Besides these ready biodegradability studies, an ultimate anaerobic biodegradability study is available, which showed that the substance is even ultimately anaerobically biodegradable with a plateau at 67 % after 79 days (ISO 11734: 1995).