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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:
09 April 2019 to 22 August 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Light yellow liquid
- Storage conditions: Ambient temperature

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
TEST INOCULUM
- Activated sludge was collected from the Easton Wastewater Treatment Facility, Easton, Maryland on 09 April 2019.
- The Easton facility treats predominantly residential wastes.
- The sludge was sieved using a 2-mm screen and adjusted to approximately 1000 mg total suspended solids/L with mineral media (see Appendix I, attached) and then aerated at test temperature until use.
- A total suspended solids measurement and standard plate count were performed on the inoculum on the day of use in the test.
- Plates were incubated at 20 ± 3 °C for approximately 48 hours.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
other: carbon
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
OBJECTIVE
- The objective of the study was to measure the amount of carbon dioxide (CO2) produced from the biodegradation of a nonvolatile organic test substance.
- This value is expressed as a percentage of the theoretical amount of CO2 (TCO2) that could have been produced if complete biodegradation of the test substance to CO2 and other inorganic constituents occurred.

EXPERIMENTAL DESIGN
- The test contained a blank control group, a reference group, a treatment group and a single toxicity control. Each group contained three replicate test chambers. The control group was used to measure the background CO2 production of the inoculum and was not dosed with a carbon source.
- The reference group chambers were used to check the viability of the inoculum and were dosed with sodium benzoate, a substance known to be biodegradable, at a nominal concentration of 10 mg C/L.
- The treatment group chambers were used to evaluate the test substance at a nominal concentration of 10 mg C/L.
- The toxicity control was used to evaluate toxicity of the test substance to the inoculum and was dosed with both the reference (10 mg C/L) and test (10 mg C/L) substances.

REFERENCE SUBSTANCE
- A stock solution of the reference substance was prepared in GenPure water at a nominal carbon concentration of 400 mg C/L. GenPure is a trademarked water purification system that produces Type I ultrapure water with a resistivity of approximately 18.2 MΩ.cm.
- The stock solution was analysed for total organic carbon (TOC). The reference substance was administered to the reference group and toxicity control test chambers by volumetric addition.
- Dosing amounts were based on the measured carbon content of the reference substance solution as determined using a Shimadzu Model TOC-VCSH carbon analyser.

TEST MEDIUM
- The test medium was a modified biochemical oxygen demand (BOD) test solution and was prepared using high quality water as described in the study protocol.
- All chemicals and reagents used in the preparation of the test medium were reagent grade or better.

TEST APPARATUS AND CONDITIONS
- The test chambers were amber 4 L bottles. The air entering the chambers was passed through Drierite to remove ambient moisture and then through Ascarite to produce CO2-free air. The air exiting the test chambers was passed through a series of three gas washing bottles, each containing approximately 100 mL of 0.5 M potassium hydroxide (KOH) to trap the CO2 that had evolved within the chamber. An additional set of gas washing bottles that was not connected to a chamber was maintained concurrently with the traps connected to the chambers. These bottles contained approximately 100 mL of 0.5 M KOH and the amount of CO2 detected in the KOH solution was subtracted from the CO2 in the control traps to determine the amount of CO2 produced by the inoculum in the blank control.
- The test was conducted at 20 ± 3 °C. Room temperature was monitored continuously using an automatic data logger.
- Test chambers were identified by project number, test substance ID, test concentration and vessel number. Magnetic stir bars and stir plates were used to mix the contents of the test chambers. Stir plate motors were cycled on and off approximately every 15 minutes to prevent the heating of the stirrer motors.

CARBON ANALYSIS
- The measured total organic carbon (TOC) concentration of stock solution for the reference substance was 398.8 mg C/L. The volume of stock solution used to dose the test, reference and toxicity control chambers was adjusted based on the measured TOC value of the appropriate solution so that approximately 10 mg C/L was delivered.

PREPARATION OF TEST CHAMBERS
- The following were added to each chamber:
1) 2470 mL of high grade water
2) 3 mL calcium chloride solution (2.75%)
3 mL of ferric chloride solution (0.025%)
3 mL of magnesium sulfate solution (2.25%)
30 mL of phosphate buffer (pH 7.4)
3) A volume of activated sludge soil-amended inoculum to achieve a final TSS concentration
of ≤ 30 mg/L
- All chambers were aerated with CO2-free air for approximately 24 hours at a rate of approximately 55 mL per minute to purge the systems of CO2. After the aeration period, the flow of CO2-free air was stopped and three CO2 traps, each containing approximately 100 mL of 0.5 M KOH, were connected to the exit air lines of each chamber.
- Reference substance stock solution (75.2 mL) was added to the appropriate chambers to achieve a nominal concentration of 10 mg C/L.
- Test substance was weighed out (0.0630 g) and added to the appropriate chambers to achieve a nominal concentration of 10 mg C/L.
- The final volume within all chambers was brought up to 3000 mL by the addition of GenPureTM water.

BIODEGRADATION TEST INITIATION
- The biodegradation test was started by bubbling CO2-free air through each of the test chambers at a rate of approximately 55 mL per minute.
- The CO2 produced from the degradation of organic carbon sources within the test chamber was trapped as potassium carbonate (K2CO3) in the KOH solution and the amount of inorganic carbon in the trapping solution was measured at various intervals during the study, using a Shimadzu Model TOC-VCSH carbon analyser.

CALCULATIONS
- The results of the inorganic carbon analyses of the CO2 traps were converted to mg CO2 produced
using the equation mg CO2 = result (mg C/L) * vol. of KOH (L) * 3.67 mg CO2/mg C
- The cumulative mg of CO2 values for the test and reference substances were corrected for the
amount of CO2 evolved by the control using the equation Cumulative mg CO2 produced = Σ mg CO2 test - mean Σ mg CO2 blank control
- The percentage of theoretical carbon dioxide (%TCO2) evolved was calculated using the equation %TOC = [mg CO2 evolved / (mg of carbon in test) x (3.67 mg CO2/mg Carbon)] * 100
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
Sigma Aldrich (CAS number 532-32-1; lot SLBW2610; purity 100 %; retest date November 2021)

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
3.3
Sampling time:
28 d
Details on results:
OBSERVATIONS AND MEASUREMENTS
- The temperature range recorded during the test was 19.15 to 21.60 ºC and was within the protocol specified range throughout the test. The results of the standard plate count and total suspended solids (TSS) measurement performed on the inoculum were 85 × 10E+03 colony forming units (CFU) per mL and 970 mg/L, respectively.
- The measured dissolved organic carbon (DOC) on Day 29 and pH values of the test chamber contents on Day 28 are presented in Table 1 (attached).
- The measured concentrations of inorganic carbon in the carbon dioxide trapping solutions are presented in Table 2 (attached).
- The cumulative amounts of CO2 produced over the test period are presented in Table 3 (attached).
- The cumulative percent of theoretical carbon dioxide (% TCO2) evolved is presented in tabular and graphical forms in Table 4 (attached) and Figure 1 (attached).
- The blank control chambers evolved an average of approximately 79 mg CO2 over the test period, corresponding to 26 mg CO2/L. This value has been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, even when freshly prepared, contains carbonates. The amount of CO2 evolved by the blank control chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO2 evolution tests.
- The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which an average of 88.1 % of theoretical CO2 was evolved over the test period (see Table 4, attached).
- An average percent biodegradation of greater than 60 % was achieved by Day 9, thereby fulfilling the criteria for a valid test by reaching the pass level by Day 14 for the reference substance.
- The final mean percent biodegradation for test item was 3.3 % by the end of the test.
- The test item may not be considered readily biodegradable since the pass level of 60 % TCO2 was not achieved by day 28 of the test.
- The toxicity control achieved > 25 % degradation by Day 14 and therefore the test item may be considered non-inhibitory at the concentration tested in this study.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Evidence of ready biodegradability in a Carbon Dioxide Evolution Test is 60 % TCO2 within the 28-day test period. In addition, the pass level must be reached within 10 days of achieving 10 % TCO2. The final mean percent biodegradation for the test item was 3.3 % by the end of the test. A negative result in a test of ready biodegradability does not necessarily mean that the test substance will not be biodegradable under relevant environmental conditions, but that additional testing maybe needed. The toxicity control achieved > 25 % degradation by Day 14 and therefore the test item may be considered non-inhibitory at the concentration tested in this study.
Executive summary:

GUIDELINE

The study protocol was based on the procedures specified in the OECD Guideline for Testing of Chemicals, Method 301B and Commission Directive, Annex V, 92/69/EEC, Method C.4-C, Carbon Dioxide (CO2) Evolution. Tests of ready biodegradability are stringent tests that provide limited opportunity for acclimation and biodegradation to occur.

 

METHODS

In the CO2 test, inoculated mineral medium was dosed with a known amount of test substance as the nominal sole source of organic carbon and aerated with CO2-free air. The CO2 produced from the mineralization of organic carbon within the test chambers was displaced by the flow of CO2-free air and trapped as K2CO3 in KOH trapping solution. The amount of CO2 produced by the test substance (corrected for that evolved by the blank inoculum) is expressed as a percentage of the theoretical amount of CO2 (TCO2) that could have been produced if complete biodegradation of the test substance occurred. The test contained a blank control group, a reference group and a treatment group, each with three replicates and a single toxicity control. The blank control was used to measure the background CO2 production of the inoculum and was not dosed with a carbon source. The reference chambers were dosed with sodium benzoate, a substance known to be biodegradable, at a nominal concentration of 10 mg C/L. The treatment group test chambers were used to evaluate the test item at a nominal concentration of 10 mg C/L. The toxicity control was used to evaluate the toxicity of the test substance to the inoculum and was dosed with both the reference (10 mg C/L) and test substances (10 mg C/L).

 

RESULTS

Results indicated that the activated sludge inoculum was active, degrading the reference substance an average of 88.1 % by the end of the test and that the test substance was not inhibitory to the inoculum at the concentration tested, as the toxicity control exceeded 25 % degradation by Day 14 of the study. The average cumulative percent biodegradation for the test item was 3.3 % by the end of the test.

 

CONCLUSION

Evidence of ready biodegradability in a Carbon Dioxide Evolution Test is 60 % TCO2 within the 28-day test period. In addition, the pass level must be reached within 10 days of achieving 10 % TCO2. The final mean percent biodegradation for the test item was 3.3 % by the end of the test. A negative result in a test of ready biodegradability does not necessarily mean that the test substance will not be biodegradable under relevant environmental conditions, but that additional testing maybe needed. The toxicity control achieved > 25 % degradation by Day 14 and therefore the test item may be considered non-inhibitory at the concentration tested in this study.