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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2018-11-12 to 2019-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 07 November 2018.
- Storage length: five days before the inoculation of mineral medium, six days before test item administration (start of the test).
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. 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 5 g dry material per litre was mixed with test water (mineral medium) and then aerated under test conditions (for 5 days) until use.
- pH: The pH of the activated sludge inoculum after preparation was 7.63. A pH adjustment of the activated sludge inoculum was not performed.
- Concentration of sludge: 4040 g suspended solids/L
- Water filtered: yes
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
test mat.
Initial conc.:
11 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
Composition of medium: Mineral medium (prepared according to OECD 301) was used.
- Test temperature: 20.1 - 21.5 °C
- pH: 7.53 - 7.83
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: 25 g dry material/L

TEST SYSTEM
- Culturing apparatus: 1500 mL flasks
- Apparatus blanks: 2, containing mineral medium only
- Test flask: 2, containing test item, mineral medium and inoculum
- Measuring equipment: All TC and IC measurements were performed with Schimadzu Total Organic Carbon Analyzer (TOC-L CSH type), the TOC and DOC are extrapolated values.
- Apparatus
Carbon content analyser: Shimadzu Total Organic Carbon; Analyzer (TOC-L CSH type); No: H54425200311
Balances: Sartorius ED 224S; No.: 33306587; Mettler Toledo AB54-S; No.: 1122092721
Water purification system: MILLIPORE Synergy UV HF ASTM 1: F8JA80461C
Centrifuge: HERAEUS Megafuge 40R; No.: 41210290
- Used Materials:
Potassium phthalate monobasic: SIGMA-ALDRICH Batch No.: BCBM6069V, Retest Date: December 2019
Sodium hydrogen carbonate: REANAL (lach:ner) Batch No.: PP/2016/04665, Expiration date: 28 February 2020
Sodium carbonate: SIGMA-ALDRICH Batch No.: SZBF3610V, Retest Date: 09 June 2019
Ultrapure water: ASTM Type 1, Prepared by MILLIPORE Synergy UV HF ASTM 1 system, Millipore

SAMPLING:
- Determinations of Carbon Content (Total, Inorganic, Total Organic, Dissolved Organic):
- Sampling method: The samples were taken from inoculum flasks, procedure control flask and test item flask. At the start of the test the total carbon (TC) and inorganic carbon (IC) content was measured in the test item suspension in mineral medium from samples, from the inoculum control flasks and from the procedure control flask. The total organic carbon content (TOC) of the samples was extrapolated.
The appropriate portion of the samples were centrifuged at 4000 g for 15 minutes. The supernatant phase was decanted and measured for TC and IC contents. The above centrifugation step (sample preparation) allowed the dissolved organic carbon (DOC) content analysis of each sample.
At the end of the test TC, IC and TOC-DOC contents of the samples taken from the same flasks were determined. The TOC-DOC analysis of samples served primarily the verification of the reference item content (on DOC basis) of appropriate test flasks; gave information about the test item solubility, test item content; furthermore, provided additional information about the biodegradation processes.
- Sampling frequency: on days 0, 2, 4, 8, 10, 14, 18, 23 and 29

- Calculated ThCO2 of the Test Item: For the calculation of the ThCO2 of the test item the molecular formula of C17H19ClO7 and the molecular weight of 370.8 g/mol g were considered.
When taking the carbon content of test item into consideration, from 370.8 g test item 17*44 g CO2 can be produced (based on following equation: C+O2=CO2). From 1 mg test item 2.02 mg CO2 can be produced.


CONTROL AND BLANK SYSTEM
- Inoculum blank (blank controls): 2, containing mineral medium and inoculum
- Abiotic sterile control: 1, containing test item, mineral medium and HgCl2
- Toxicity control: 1, containing test item, positiv control, mineral medium and inoculum
- Positiv control: 2, containing reference substance, mineral medium and inoculum
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The pre-experiments on solubility of the test item and a toxicity test were not performed in compliance with the GLP-Regulations. Based on the available molecular formula of the test item, the required nominal 10-20 mg TOC/L concentration corresponds to 18.2-36.4 mg test item/L concentration.
In the frame of the preliminary solubility test the test item solubility was investigated in the concentration range of 300-20 mg/L in mineral medium. At the concentration choice a technically feasible, possible test item administration was taken into consideration. At the concentration of 20 mg/L after about 30 min. ultrasonic treatment an apparently homogeneous solution was obtained (at higher concentrations the test item could not be suspended, mixed in mineral medium).
In the preliminary toxicity test the test item was investigated at the nominal concentration of 30 mg test item/L mineral medium (that corresponds to 16.5 mg TOC/L), and at the inoculum concentration of 25 mg suspended solids/L. The viable cell count of inoculum was checked 6-times during the 28-day incubation period by sampling (1 mL) and plating 0.1 mL of the adequately diluted cultures on nutrient agar plates (two plates for each dilution level). In the toxicity test the test item was directly added to the test mixtures.
At this concentration level a slight inhibitory, toxic effect of the test item on the applied microflora was noticed; therefore, in the main experiment the investigation of slightly lower nominal concentration level (nominal: 20 mg/L) was considered.
Test performance:
The study is considered valid because:
- The Inorganic Carbon content of the test item containing test suspension (inoculated mineral medium and test item) at the beginning of the test (3.8 %) was lower than 5 % of the Total Carbon.
- The total CO2 evolution in the inoculum blank at the end of the test (on average 32.13 mg, corresponding to 10.71 mg/L medium) did not exceed 40 mg/L medium.
- The difference of duplicate values for the degradation at the plateau or at the end of the test was not greater than 20 %.
- The percentage degradation of the reference item reached the level of ready biodegradability (> 60 %) on exposure day 8. The percentage degradation of the reference item was 65.00 % on day 8.
- In the toxicity control, containing both the test item and reference item the biodegradation was higher than 25 % based on ThCO2 on day 14. The biodegradation in the toxicity control was 26.02 % within 6 days.
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
based on ThCO2
Value:
28.78
Sampling time:
28 d
Details on results:
- Calculation Based on the Evolved CO2 Measurement: Under the test conditions the percentage biodegradation of the test substance reached a mean of 28.78 % after 28 days based on its ThCO2. The highest biodegradation value of 28.81 % was noticed on the 21th day of the test. The biodegradation curve reached its plateau on the 10th day of the test and from that the slight changes in biodegradation values reflect the biological variability of the test. The pass level for ready biodegradability is the removal of 60 % ThCO2 in a 10-day window. The obtained biodegradation value remained far below the pass level; therefore, the test item was considered to be not readily biodegradable.
- Calculation Based on the DOC Measurements: At the start of the test the total carbon (TC) and inorganic carbon (IC) content was measured in the test item suspension in mineral medium from samples, from the inoculum control flasks and from the procedure control flask . The total organic carbon content (TOC) of the samples was extrapolated. The appropriate portion of the samples was centrifuged at 4000 g for 15 minutes, and the supernatant phase decanted and measured. The TC and IC contents of supernatant phase allowed the dissolved organic carbon (DOC) content analysis of each sample. At the end of the test the same measurements and procedures were performed and TOC and DOC contents of the samples (originated from the same test mixtures) were determined.
- The time between test item treatment and sampling (technically feasible, short, ~10 minutes mixing of test suspension with magnetic stirrer, before complete closing the aeration system) did not allow to reach satisfying dissolution or homogeneity of the test item suspension, that is was reflected in the obtained low DOC value: at the start of the test the DOC content of test item containing mixture after inoculum control correction was 1.222 mg/L, that is 11 % of the nominal (the test item was added in nominal concentration of 20 mg/L that corresponds to nominal 11 mg organic carbon /L).
- Abiotic Control: In the abiotic control containing the test item and sterilized inoculum 1.10 % degradation was noted after 28 days within the biological variability range of the applied test system). The calculated abiotic degradation values did not show increasing tendency.
- Correction of test item biodegradation value with abiotic processes was considered as not necessary.
- Biodegradation of Toxicity Control: In the toxicity control containing both, the test item and the reference item, a mean of 26.02 % biodegradation was noted within 6 days and 47.77 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred already within 6 days).
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 65.00 % already after 8 days, and to 82.81 % after 28 days of incubation, based on its ThCO2, thus confirming the suitability of the used activated sludge inoculum.
Based on the supplemental total carbon (TC), inorganic carbon (IC) analyses and derived DOC contents of the samples taken from the reference item containing suspension at the start and end of the test higher, about 92 % biodegradation was calculated.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the test conditions used, the percentage biodegradation of the test substance reached a mean of 28.78 % after 28 days based on its theoretical carbon dioxide (ThCO2). In conclusion, the test item
was considered to be not readily biodegradable. A 10-d-window could not be determined during the 28-day test.
Executive summary:

The ready biodegradability of the test item was determined in a CO2 Evolution Test (Modified Sturm Test) under GLP conditions according to OECD 301 B (1992). A defined volume of inoculated mineral medium, containing a known concentration of the test item as the nominal sole source of organic carbon (in the range of 10-20 mg/L dissolved organic carbon: DOC or total organic carbon: TOC) was aerated by carbon dioxide-free air at a controlled rate in the dark. The degradation was followed over 28 days by determining the carbon dioxide produced. The CO2 was trapped in barium hydroxide and was measured by titration of the residual hydroxide. The amount of carbon dioxide produced from the test substance (corrected for that derived from the blank inoculum) is expressed as percentage ThCO2. The degree of biodegradation was also calculated from the supplemental TOC and DOC analysis made at the start and end of incubation. The chosen test item concentration of 20 mg/L (that corresponded to the nominal of 11 mg TOC/L) investigated in the main test was based on the results of the preliminary solubility test and based on the preliminary information about the toxicity of the test item. In parallel (under the same conditions as the test item), inoculum control, reference item, sodium benzoate at the concentration of 17.14 mg/L (that corresponded to the nominal of 10 mg DOC/L) as procedure control), abiotic control and toxicity control were investigated. In this study all validity criteria regarding the inoculum, procedure and toxicity controls, inorganic carbon (IC) content of test item suspension, and biodegradation values in the test item parallels were met. Under the test conditions used, the percentage biodegradation of the test item reached a mean of 28.78 % after 28 days based on its theoretical carbon dioxide (ThCO2) which is less than the pass level for ready biodegradability of 60 %. The test item biodegradability reached its plateau on about the 10th day. The highest biodegradation value of 28.81 % was noticed on the 21th day of the test. The slight changes in biodegradation values reflect the biological variability of the test.

Description of key information

The substance is not readily biodegradable according to OECD 301 B.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Under the test conditions used in a study, the percentage biodegradation of the test substance reached a mean of 28.78 % after 28 days based on its theoretical carbon dioxide (ThCO2). The test item  was considered to be not readily biodegradable according to OECD 301 B (1992). A 10-d-window could not be determined during the 28-day test.