Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

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:

2011-01-10 to 2011-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Test System
Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The activated sludge used for this study was washed by centrifugation (10 min) and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged (10 min). This procedure was repeated three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water and then aerated until use. Before use the sludge was filtered through cotton wool.

Duration of test (contact time):

28 d

Initial conc.:

2.3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test Units
Type and Size
BOD bottles of 300 ml volume with special neck and glass stoppers.
Identification
Each of the test flasks were uniquely identified with study code, test group, days of measurement and replicate number.
Test Conditions
Surrounding Type
Incubator and controlled environment room (during the formulation and oxygen measuring)
Temperature: 20.0 – 22.4°C
Light Conditions: Darkness
pH-Value of Reconstituted Water:
7.23 (measured without adjustment at the start of the test)
Oxygen conc. of Reconstituted Water:
9.0 mg O2/L (measured at the start of the test)
Recording:
Test conditions were measured with suitable instruments and documented in the raw data.
Reconstituted Water:
In deionised water analytical grade salts were added to prepare the following stock solutions:
a) 2.125 g KH2PO4, 5.4375 g K2HPO4, 16.795 g Na2HPO4 x 12H2O, 0.125 g NH4Cl filled up with deionised water to 250 mL volume
b) 5.625 g MgSO4 x 7H2O filled up with deionised water to 250 mL volume
c) 9.10 g CaCl2 x 2H2O filled up with deionised water to 250 mL volume
d) 0.25 g FeCl3 x 6H2O filled up with deionised water to 1000 mL volume (stock solution was applied on the day of preparation)
Ratio of ingredients:
1 mL of the stock solutions a) - d) were combined and filled to a final volume of 1000 mL with deionised water. The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was about 9.0 mg/L at about 20 °C.

Equipments:
Large glass tanks with valve,
Large glass bottles with valve,
Narrow necked,300 mL BOD bottles with glass stoppers,
Funnels and coarse filter papers,
Moisture Content Analyzer,
Centrifuge, Balance,
Self stirring O2 electrode,
Oxygen and pH meter,
Aeration system,
Incubator with thermometer

Study Design
Preparation of the Test Solutions
The components were applied in the following ratio in the test flasks:
The preparation of the respective test solutions with Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was performed according to the followings: The respective amount of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was weighed in directly to reach the required test item concentration of 2.3 mg/L. During the performance of the test the test solutions were ultrasonicated under continuous cooling for 5 min. to ensure a good dispersion. The chosen test item concentration was based on the measured chemical oxygen demand (COD): 2.61 ±0.04 mg O2/mg test item and on the performed 14-d preliminary test.
Test Item (flasks 1a and 1b):
Based on the chemical oxygen demand (COD) of 2.61 mg O2/mg test item, 8.0 mg of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was thoroughly mixed into 3.48 litres of aqueous test medium (corresponding to 2.3 mg/L test item, respectively a COD of about 6.003 mg O2/L).
Procedure Control: Sodium benzoate (flasks 2a and 2b):
Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg) (calculation based on elemental composition and molecular weight; details are given in the guidelines), stock solution* corresponding to 12.096 mg of Sodium benzoate was mixed into 3.36 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L). * the concentration of the stock solution was 360 mg/L
Inoculum Control (flasks 3a and 3b):
Only filtered inoculum was added to 3.40 litres of aqueous test medium.
Toxicity Control (flasks 4a and 4b):
8.0 mg of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc and reference item stock solution* (34.8 mL) were mixed into 3.48 litres of aqueous test medium corresponding to 2.3 mg/L test item (COD of 6.003 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L). * the concentration of the stock solution was 360 mg/L
General:
Microbial inoculum (0.5 ml per litre) was added to each preparation bottle.
Course of the Test
Preparation of Test Flasks
Sufficient number of BOD flasks was cleaned with 5-10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly stoppered.
The following bottles were prepared:
10 bottles containing the test item and inoculum
10 bottles containing the reference item and inoculum (procedure control)
10 bottles containing only inoculum (inoculum control)
10 bottles containing the test item, reference item and inoculum (toxicity control)
Incubation Period: 28 days
Test Parameters
Measurement of Oxygen:
The oxygen concentrations were measured with oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.
Measurement of Temperature: Temperature was measured continuously and registered on weekdays.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Due to the poor solubility of test substance a 14d-preliminary study was performed. Based on the results obtained, the test solutions were ultrasonicated under continuous cooling for 5 min during performance of the test in order to ensure a good dispersion. The chosen test item concentration was based on the measured chemical oxygen demand (COD): 2.61 ±0.04 mg O2/mg test item

Test performance:

The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

Parameter:

% degradation (O2 consumption)

Value:

15

Sampling time:

28 d

Details on results:

- Biodegradation of Test Item
Percentage Biodegradation:
Under the test conditions the percentage biodegradation of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc reached a mean of 15.0 % after 28 days based on the measured COD of the test item.
Conclusion:
The test item can be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % COD.

Parameter:

COD

Value:

2.61 g O2/g test mat.

Results with reference substance:

- Biodegradation of Reference Item
Percentage Biodegradation:
The reference item Sodium benzoate was sufficiently degraded to a mean of 83.3 % after 14 days, and to a mean of 90.0 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.
Conclusion:
The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.


- Biodegradation of Toxicity Control
Percentage Biodegradation:
In the toxicity control containing both, the test item and the reference item, a mean of 40.4 % biodegradation was noted within 14 days and 43.6 % biodegradation after 28 days of incubation.
Conclusion:
According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the test conditions the percentage biodegradation of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc reached a mean of 15.0 % after 28 days based on the measured COD of the test item. Thus, the test item can be considered as not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % COD.

Executive summary:

Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was tested for ready biodegradability in a Closed Bottle test according to EU method C.4-E and OECD guideline 301 D. Under the test conditions the percentage biodegradation of the test substance reached a mean of 15.0 % after 28 days based on the measured COD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % COD. The 10d-window started on day 21, degradation was below the pass level of 60 % based on oxygen consumption at the end of the test. Thus, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc can be considered as not ready biodegradable.

Description of key information

Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was tested for ready biodegradability in a Closed Bottle test according to EU method C.4-E and OECD guideline 301 D. The test item was considered not readily biodegradable. The degree of biodegradation reached 15 % after 28 days. The 10d-window started on day 21, degradation was below the pass level of 60 % based on oxygen consumption at the end of the test. 

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was tested for ready biodegradability in a Closed Bottle test according to EU method C.4-E and OECD guideline 301 D. Under the test conditions the percentage biodegradation of the test substance reached a mean of 15.0 % after 28 days based on the measured COD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % COD. The 10d-window started on day 21, degradation was below the pass level of 60 % based on oxygen consumption at the end of the test. Thus, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc can be considered as not ready biodegradable.