Enzymatic hydrolyis products of Candida Saitoana

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:

December 2018 - January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Enzymatic hydrolyis products of Candida Saitoana

EC Number:

952-608-9

IUPAC Name:

Enzymatic hydrolyis products of Candida Saitoana

Test material form:

solid: particulate/powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Sludge collection: a fresh sample of activated sludge was collected on Dec 27th, 2018 from the aeration tank of the Tian shan Wastewater Treatment Plant in Shanghai which treats predominantly domestic sewage with Anacrobic-Oxic process (A/O). The activated sludge was kept by aeration until used(non-aeration during transport from the wastewater treatment plant to the laboratory was kept to a minimum).
Sludge treatment: When back to the laboratory, the sludge was washed with mineral medium. After centrifugation, the supernatant was decanted. This procedure was repeated three times(4000 rpm, 4°C, centrifuge 20 minutes). Then, 0.401g of the concentrated sludge was weighed, dried at 105°C for 1h and then measured by moisture meter for 1h to determine the dry weight. The dry weight of concentrated sludge was 9.73%.
Sludge suspension: According to the dry weight, 30.8g of concentrated sludge was calculated and suspended in 1L mineral medium to yeild a concentration of 3 g suspended solids/L. The final concentration of the activated sludge in the test medium was 30mg/L.

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 47.8 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test substance was soluble according to the information supplied by the sponsor. To prepare the stock solution of test substance, 250.02mg of test substance was dissolved in 500mL mineral medium and the concentration of stock solution was 500mg/L (see in 13.3).
To prepare the stock solution of reference substance, 1000.07mg of sodium benzoate was dissolved in 1L mineral medium and the concentration of stock solution was 1g/L. Final concentration of reference substance in test system was 100mg/L.
Respirometer operation
When the test system was prepared completely, Respirometer (WTW, OxiTop 110C, Weilheim, Germany) Operational procedure was performed as follows:
1) Evaluate the BOD range of the test substance and prepare the test systems according to the preparation table in 13.3.
2) Insert a magnetic stirrer bar into the flasks.
3) Place 2 sodium hydroxide pellets in the rubber sleeve.
4) Insert the rubber sleeves into the flasks.
5) Screw the OxiTop® measuring head tightly.
6) Start the measurement on the OxiTop® head.
7) Place the measuring flask on the inductive stirring system, then set the temperature of the incubator at 22°C and the temperature fluctuation was 21.9-22.3°C during 28 days, in the dark.
8) Auto-read the results once per 112min during 28 days.
9) Determine pH at the beginning and the end of the study.
10) The concentration of nitrite and nitrate are analysed by using Hach NitraVer 5 Nitrate kit and Hach NitriVer 3 Nitrite kit at the end of test, respectively.
Determination of Nitrification
Since the test substance contains nitrogen, nitrification with formation of nitrite and/or nitrate may occur during biodegradation of the test substance. 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 substance and in the inoculums controls (Hach NitraVer 5 Nitrate kit and Hach NitriVer 3 Nitrite kit). Prior to measurement, the samples were filtered through a 0.45μm nitrocellulose membrane (Millipore, pore size 0.45μm).
The nitrite concentration in the measured test flasks containing the test substance were 0.0042mg NO2-N/L and 0.0038mg NO2-N/L. The nitrite concentration of TS were close to the mean concentration of IB (0.0041mg NO2-N/L). The nitrate concentration in the measured test flasks containing the test substance were 1.1mg NO3-N/L and 1.2mg NO3-N/L. The nitrate concentration of TS were not greater than the mean concentration of IB (1.2mg NO3-N/L).
Therefore, the nitrification process can be neglected, so the correction for nitrification was unnecessary.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

none

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 67

Sampling time:

28 d

Details on results:

Results showed that under the experimental conditions, biodegradation of test substance attained an average of 63% at Day 12 (10-d window) and 67% at Day 28 which met the 10-d window criteria.
Therefore, the test substance can be considered to be readily biodegradable under the experimental conditions according to this test method.

The mean BOD of the inoculum blanks was 24.05mg O2/L in 28 days, which was less than 60 mg/L, meeting the guideline requirement. During the 28 days, the pH values of the solution in test substance flasks were in the range of 7.40-7.71, which met the requirement of pH6.0-8.5 of the test substance solution. The difference between replicate values of the removal of the test substance during 28d test period was less than 20%.
The mean biodegradation of the reference substance (Sodium benzoate) was 89% by Day 14, reaching the pass level of the ready biodegradation test (>60% within 14 days).
The biodegradation percent of the toxicity control was 87% by day 14, ≥25% based on total ThOD, which met the guideline requirement.
Thus, the test is considered valid.

BOD5 / COD results

Results with reference substance:

The percent of biodegradation in the toxicity control, containing both the test substance and reference substance, was calculated based on the sum of the ThODNH3 of the test substance and the ThODNH3 of the reference substance.
In the toxicity control, within 14 days of exposure, biodegradation amounted to 87%. Since biodegradation in the toxicity control was greater than 25% within 14 days, the test substance was considered not to have a toxic or inhibitory effect on the activity of the microbial inoculum.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

All the validity criteria for OECD 301F were successful.
The test substance was readily biodegradable under the present test conditions.

Executive summary:

The study was conducted according to the guidelines of “Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals-Degradation and Accumulation, 2013, 301F Ready Biodegradability: Manometric Respirometry Test” and “OECD, Guidelines for the Testing of Chemicals. Ready Biodegradability 301F: Manometric Respirometry Test, 1992”.

Under specific experimental conditions, the ready biodegradability of the test item was determined in a 28-day dissolved oxygen depletion test using activated sludge from a domestic waste water treatment plant.tions, the ready biodegradability of the test item was determined in a 28-day dissolved oxygen depletion test using activated sludge from a domestic waste water treatment plant.

The tested concentration of test substance was 47.80mg/L (i.e.50.67mgThOD_NH3/L). The concentration of sludge inoculum in test system was 30mg/L, and the tested concentration of sodium benzoate used as reference substance was 100 mg/L (i.e.167.00mg ThOD_NH3/L). A toxicity control was also included. 

During the test, the temperature was maintained at 21.9-22.3°C. The mean total O₂uptake in the inoculum blanks at the end of the test was less than 60mg O₂/L in 28 days meeting the guideline requirement.Biodegradation of the reference substance (sodium benzoate)reached the pass level of the ready biodegradation test(>60% within 14 days).

Biodegradation of the reference substance (sodium benzoate) reached an average of 89% by Day 14. The difference of extremes between replicate values of the removal of the test substance during the 28d test period was less than 20%. The results of the toxicity control showed that the test substancemet the criteria for not being inhibitory to the microbial inoculum. Thus, the test is valid.

Results showed that under the experimental conditions, biodegradation oftest substanceattained an average of 63% at Day 12(10-d window). Biodegradation of test substance attained an average of 67% at Day 28 which met the 10-d window criteria.

Therefore,the test substancecanbe considered to be readily biodegradable under theexperimentalconditions according to this test method.