Registration Dossier

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:
2009-02-24 to 2009-06-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform astudy under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Test material form:
solid
Details on test material:
TEST ITEM
The test item and the following information were provided by the Sponsor:
Chemical Name: Reaction product of amino acids and palmitoyl chloride
Identity: LCE08130 Batch No.: 0826900021
Purity: Not available; therefore, excluded from the Statement of Compliance.
Expiration Date: 25-Sep-2011 (retest date)
Solubility in Water: Partly miscible, slightly soluble (<<0.5 g/L)
Aggregate State / Physical Form at Room Temperature: Solid (flakes)
Storage Conditions: At room temperature at about 20 °C, away from direct sunlight.
Specific details on test material used for the study:
TEST ITEM
The test item and the following information were provided by the Sponsor:
Chemical Name: Reaction product of amino acids and palmitoyl chloride
Identity: LCE08130 Batch No.: 0826900021
Purity: Not available; therefore, excluded from the Statement of Compliance.
Expiration Date: 25-Sep-2011 (retest date)
Solubility in Water: Partly miscible, slightly soluble (<<0.5 g/L)
Aggregate State / Physical Form at Room Temperature: Solid (flakes)
Storage Conditions: At room temperature at about 20 °C, away from direct sunlight.

REFERENCE ITEM
The reference item and the following information were supplied by Fluka (BioChemika):
Identity: Sodium benzoate Product No.: 71295
Lot No.: 1348422 Purity: 99.8%
Expiration Date (as given by the supplier): Jun-2013 (date of recommended retest)
Expiration Date (handled at Harlan Laboratories): 30-Jun-2013 (date of recommended retest)
Storage Conditions: In tightly closed original container, at room temperature at about 20 °C.

Study design

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): The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater.
- Laboratory culture: not applicable
- Method of cultivation: not applicable
- Storage conditions: During the holding period the sludge was aerated at room temperature.
- Storage length: three days prior to use
- Preparation of inoculum for exposure: Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis).
- Concentration of sludge: Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.
- Initial cell/biomass concentration: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter.
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
50 mg/L
Based on:
test mat.
Initial conc.:
102.5 mg/L
Based on:
COD
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:
1) KH2PO4 8.50 g/L; K2HPO4 21.75 g/L; Na2HPO4 x 2H2O 33.40 g/L; NH4Cl 0.50 g/L. The pH of this solution was 7.4.
2) MgSO4 x 7H2O 22.50 g/L
3) CaCl2 x 2H2O 36.40 g/L
4) FeCl3 x 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter
To obtain the final test water, 10 mL of stock solution No. 1 and 1 mL each of stock solution Nos. 2, 3 and 4 were combined and made up to 1000 mL with purified water. The pH was adjusted from 7.7 to 7.4 with a diluted hydrochloric acid solution.
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: about 20 °C
- pH: at test start 7.4: at test end 7.5 - 8.2
- pH adjusted: yes (test water only)
- CEC (meq/100 g): no data
- Aeration of dilution water: not reported
- Suspended solids concentration: amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: SAPROMAT D12 (Voith GmbH, Heidenheim, Germany)
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: stirring; replenishing of consumed oxygen in haed space of test vessels via electrolytical dissociation of copper sulphate solution
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: The CO2 is adsorbed by soda lime, which results in a decrease of the total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer.
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: soda lime particles are placed in a basket, which is inserted in the head space of the air tight reaction vessel.

SAMPLING
- Sampling frequency: The CO2 is adsorbed by soda lime, which results in a decrease of the total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer (process runs every minute)
- Sampling method: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: activated sludge, test medium, without test item or reference item
- Abiotic sterile control: no
- Toxicity control: activated sludge, test medium, plus 50 mg/l test item and 100 mg/l reference item (theoretical oxygen demand 168 mg/l)
- Procedure control: activated sludge, test medium, plus 100 mg/l reference item (theoretical oxygen demand 168 mg/l)

STATISTICAL METHODS: none applied
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
no
Test performance:
The biochemical oxygen demand (BOD) of LCE08130 in the test media significantly increased from test start until test termination after 28 days. Measurement of nitrate and nitrite concentrations at the end of the test indicated that nitrification had occurred in the test media containing the test item during the test period. After correction of the oxygen consumption for the oxygen consumed during the formation of nitrate and nitrite, mean
biodegradation of LCE08130 amounted to 108%.

In the toxicity control, the run of the curve of the oxygen consumption over the 28-day exposure period was similar but significantly higher than the one of the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation amounted to 119%. After
correction for the oxygen consumed in the process of nitrification, biodegradation at the end of the test amounted to 128%.
Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 50 mg/L because biodegradation in the toxicity control was >25% within 14 days.
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
108
Sampling time:
28 d
Remarks on result:
other: Result is nitrification corrected
Details on results:
Since the test item contains nitrogen, nitrification with formation of nitrite and/or nitrate may occur during biodegradation of the test item. If nitrification occurs, the oxygen demand must be corrected for the oxygen consumed by oxidation of ammonium into nitrite and/or nitrate. Therefore, at the end of the test, the nitrite and nitrate concentrations were measured in the test media containing the test item and in the inoculum controls. Prior to measurement, the samples were filtered through a nylon filter (0.45 μm).
The concentration of nitrite in the test flasks containing the test item, and in the toxicity control was in the range 0.1 – 2.9 mg NO2--N/L. This corresponds to 0.4 – 10 mg O2/L, consumed during formation of nitrite (the oxygen consumed during nitrite formation is 3.43 multiplied by the concentration of NO2--N). The mean nitrite concentration in the flasks containing only inoculum was 0.3 mg NO2--N/L, corresponding to 1.0 mg O2/L consumed during the formation of nitrite.
The nitrate concentration in the test flasks containing the test item, and in the toxicity control was in the range 2.3 – 3.2 mg NO3--N/L, corresponding to 11 – 15 mg O2/L consumed during the formation of nitrate (the oxygen consumed during nitrate formation is 4.57 multiplied by the concentration of NO3--N). The mean nitrate concentration in the flasks containing only inoculum was 1.6 mg NO3--N/L, corresponding to 7.3 mg O2/L consumed during the formation of nitrate.
Therefore, the measured BOD in the test flasks containing the test item was corrected for the oxygen consumed by nitrification.

BOD5 / COD results

BOD5 / COD
Parameter:
COD
Value:
2.05 g O2/g test mat.
Results with reference substance:
In the procedure controls, the reference item was degraded by an average of 88% by Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 94%.

Any other information on results incl. tables

Summary data table and data plots on biodegradation of LCE08130 see attached.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The percent biodegradation of the test item LCE08130 was calculated based on the measured chemical oxygen demand (COD) of 2.05 mg O2/mg test item. Mean biodegradation of LCE08130 amounted to 108%. Consequently, LCE08130 was found to be completely biodegradable under the test conditions within 28 days.
Executive summary:

The test item LCE08130 was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to the EU Commission Directive 92/69 EEC, C.4-D Commission Regulation (EC) No 440/2008, C.4-D and the OECD Guideline for Testing of Chemicals No. 301 F. The chemical oxygen demand (COD) of the test item LCE08130 was determined according to the EU Commission Directive 92/69 EEC, C.6 following DIN 38414-S9.

COD = 2.05 mg O2/mg test item

The percent biodegradation of the test item was calculated based on the measured chemical oxygen demand (COD) of 2.05 mg O2/mg test item.

The biochemical oxygen demand (BOD) of LCE08130 in the test media significantly increased from test start until test termination after 28 days. Measurement of nitrate and nitrite concentrations at the end of the test indicated that nitrification had occurred in the test media containing the test item during the test period. After correction of the oxygen consumption for the oxygen consumed during formation of nitrate and nitrite, mean biodegradation of LCE08130 amounted to 108%.

Consequently, LCE08130 was found to be completely biodegradable under the test conditions within 28 days.

The pass level for ready biodegradability, i.e. biodegradation of at least 60% of the COD in a 10-day window within the 28-day period of the test, was reached.

In the toxicity control, containing both LCE08130 and the reference item sodium benzoate, LCE08130 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 50 mg/L.

In the procedure controls, the reference item sodium benzoate was degraded by an average of 88% by Day 14, and reached an average biodegradation of 94% by the end of the test (Day 28), thus confirming suitability of the activated sludge.