Glutens, hydrolyzates, reaction products with lauroyl chloride, sodium salts

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-02-24 to 2009-07-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform study under GLP.

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)

Specific details on test material used for the study:

TEST ITEM
Chemical name: sodium lauroyl oat protein amino acids
Test item: LCE08131
Batch: 0818600011
Purity: not available, therefore, excluded from the Statement of Compliance
Expiration date: 04-Jul-2011
Solubility in water: fully miscible
Storage conditions: at room temperature, at about 20°C, away from direct sunlight

REFERENCE ITEM
Identity: Sodium benzoate
Product n°: 71295
Lot n°: 1348422
Purity: 99.8%
Expiration date: Jun-2013
Storage conditions: In tightly closed original container, at room temperature at about 20°C.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Test system:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater.
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.
During the holding period the sludge was aerated at room temperature.
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).
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.

Duration of test (contact time):

28 d

Initial conc.:

250 mg/L

Based on:

test mat.

Initial conc.:

106 mg/L

Based on:

COD

Initial conc.:

252 mg/L

Based on:

test mat.

Initial conc.:

107 mg/L

Based on:

COD

Initial conc.:

100 mg/L

Based on:

other: reference item

Initial conc.:

167 mg/L

Based on:

ThOD

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 250 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:

benzoic acid, sodium salt

Preliminary study:

no

Test performance:

The percent biodegradation of the test item was calculated based on the measured chemical oxygen demand (COD) of 0.43 mgO2/mg test item.
The biochemical oxygen demand (BOD) of LCE08131 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 (see Section 3.7). After correction of the oxygen consumption for the oxygen consumed during the formation of nitrate and nitrite, mean biodegradation of LCE08131 amounted to 95%.
Consequently, LCE08131 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, 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 93%. After correction for the oxygen consumed in the process of nitrification, biodegradation at the end of the test amounted to 96%.
Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 250 mg/L because biodegradation in the toxicity control was >25% within 14 days.

The chemical oxygen demand (COD) of the test item LCE08131 was determined according to the EU Commission Direction 92/69/EEC, Part C.6 following DIN 384141-S9.
The duplicate values were 0.41 and 0.44 mgO2/mg test item. The mean COD was 0.43 mgO2/mg test item.

The pH measured in all flasks at the start of the test was 7.4. At the end of exposure (Day 28), pH values of 7.4-8.7 were measured.

Parameter:

% degradation (O2 consumption)

Value:

95

Sampling time:

28 d

Remarks on result:

other: Result nitrification corrected

Parameter:

COD

Value:

0.43 g O2/g test mat.

Results with reference substance:

In the procedure controls, the reference item was degraded by an average of 87% 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 88%.
The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the COD of the
test item and the ThOD of the reference item.
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 93%. After
correction for the oxygen consumed in the process of nitrification, biodegradation at the end of the test amounted to 96%.
Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of
250 mg/L because biodegradation in the toxicity control was >25% within 14 days.

Summary data table and data plots on biodegradation of LCE08131 see attached.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The percent biodegradation of the test item LCE08131 was calculated based on the measured chemical oxygen demand (COD) of 0.43 mg O2/mg test item. Mean biodegradation of LCE08130 amounted to 95%. Consequently, LCE08131 was found to be completely biodegradable under the test conditions within 28 days.

Executive summary:

The test item LCE08131 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 = 0.43 mg O2/mg test item

The percent biodegradation of the test item was calculated based on the measured chemical oxygen demand (COD) of 0.43 mg O2/mg test item.

The biochemical oxygen demand (BOD) of LCE08131 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 LCE08131 amounted to 95%.

Consequently, LCE08131 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 LCE08131 and the reference item sodium benzoate, LCE08131 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 250 mg/L.

In the procedure controls, the reference item sodium benzoate was degraded by an average of 87% by Day 14, and reached an average biodegradation of 88% by the end of the test (Day 28), thus confirming suitability of the activated sludge.

Description of key information

Test material: the liquid marketed product "Proteol OAT", batch LCE08131 which contains ca.27.5% of the solid substance "Glutens, hydrozylates, reaction products with lauroyl chloride, sodium salts".

The percent biodegradation of the test item LCE08131 was calculated based on the measured chemical oxygen demand (COD) of 0.43 mg O2/mg test item. 95 % of biodegradation were reached in 28 days within the 10 -day window, the test item was considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable