Bronopol

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:

09.03.2022-06.04.2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Inoculum (activated sludge) was collected on 07th March 2022 from the aeration tank of the wastewater treatment plant of Lambsheim, Germany. A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size of about 1 mm. Subsequently, this sludge suspension was aerated for about 2 days at 22 ±2° C with carbon dioxide free air in order to reduce the inorganic carbon content in the blank controls.

At the day of exposure, the aeration was stopped, and the sludge suspension was washed once using drinking water and was allowed to settle. After settling, the supernatant was discarded. The remaining sludge suspension was filled up once more with drinking water and the final concentration of the sludge was adjusted to 6.0 g/L dry weight. Aliquots of 7.5 mL of this suspension were added to the 2 L test vessels containing 1.5 L test medium to obtain a final activated sludge concentration of 30 mg/L dry weight.

Duration of test (contact time):

28 d

Initial conc.:

30 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
The used mineral medium complies with the test guideline OECD 301B. It was prepared as follows:

Solution A: KH2PO4 : 8.50 g
K2HPO4 : 21.75 g
Na2HPO4 × 2 H2O : 33.40 g
NH4Cl : 0.50 g
The compounds were dissolved with deionized water to 1000 mL; the pH value was adjusted to 7.4.

Solution B: CaCl2 × 2 H2O : 36.40 g
The compound was dissolved with deionized water to 1000 mL

Solution C: MgSO4 × 7 H2O : 22.50 g
The compound was dissolved with deionized water to 1000 mL

Solution D: FeCl3 × 6 H2O : 0.25 g
The compound was dissolved with deionized water to 1000 mL


- Test temperature: 22 +- 2 °C
- pH: The pH was measured only at the beginning in Test substance assay 0 after setting it up and was found to be 7.3
- pH adjusted: no
- Suspended solids concentration:
- Continuous darkness: yes
- Other:
At the day of exposure, the aeration was stopped, and the sludge suspension was washed once using drinking water and was allowed to settle. After settling, the supernatant was discarded. The remaining sludge suspension was filled up once more with drinking water and the final concentration of the sludge was adjusted to 6.0 g/L dry weight. Aliquots of 7.5 mL of this suspension were added to the 2 L test vessels containing 1.5 L test medium to obtain a final activated sludge concentration of 30 mg/L dry weight.

TEST SYSTEM
- Culturing apparatus: 2 liter glass beakers filled up to 1.5 liters
- Number of culture flasks/concentration:
blank controls: 2
reference substance: 4
inhibition control: 4
test substance: 6
abiotic control: 2

- Method used to create aerobic conditions: All traps were directly attached to an aeration unit which provides bubble aeration (about one till two bubbles per second) with CO2 free air.

- Test performed in closed vessels due to significant volatility of test substance: closed bottles were used to avoid CO2 loss due to leakage.
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used:
The bottles were then connected to 4 serial scrubbing bottles of total volume 250 mL. The first two serial scrubbing bottles directly connected to the test flasks were filled with 100 mL of 0.05 mol sodium hydroxide (NaOH) solution which act as the trap to capture the carbon dioxide evolved from the mineralization processes. A third bottle filled with 50 mL ethylene glycol to absorb volatile organic substances and a fourth bottle filled with 50 mL 0.1 M sulphuric acid for capturing the volatile alkaline substances were connected to these NaOH traps. These third and fourth scrubbing bottles were measured only at the last day of measurement.
At each time point, NaOH solution from the CO2 capture trap that is directly connected to the incubation botte was used for the calculation of the produced carbon dioxide between two consecutive sampling points. When this first trap is removed from its position for sampling, the second trap was moved forward and connected directly to the test bottles. The emptied trap for TIC sampling was again filled with fresh sodium hydroxide solution and placed into the second position.


SAMPLING
- Sampling frequency: 0, 2, 6, 9, 12, 14, 19, 22, 26, 28 day
- Sampling method: LSC measurements
- Sample storage before analysis: The samples were analyzed on the day of sampling.

Reference substance:

benzoic acid, sodium salt

Test performance:

Experiment 1 began on 02nd Feb. 2022 was failed due to slow biodegradation in reference substance assays (RS). By day 14, only 59% CO2/ThCO2 was produced in RS1 and 43% CO2/ThCO2 in RS2. Test substance assays in this experiment 1 had a mean biodegradation of 14% CO2/ThCO2 and inhibition control assay had 27% CO2/ThCO2. According to OECD 301B, the RS should have a biodegradation above 60% CO2/ThCO2 by day 14. Therefore, the first experiment was discontinued, and the study was repeated in a second experiment under the same conditions.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

20

Sampling time:

28 d

Results with reference substance:

Degree of biodegradation of the reference substance
after 14 days: 70% CO2/ThCO2

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

14C-Bronopol achieved a maximum degree of biodegradation 20 ±1% CO2/ThCO2 (mean of five replicates ±SD, one day after acidification) after an exposure period of 28 days.

The degree of biodegradation at the end of the ten-day window was approx. 16 ±1% CO2/ThCO2 (mean ±SD).

In abiotic control, about 3 ±1% CO2/ThCO2 (mean ±SD) was measured after an exposure period of 28 days.

Hence, it did not achieve the required pass level for ready biodegradability in accordance with the OECD Guideline 301B i.e., at least 60% of the theoretical CO2 production must be attained within 10 days of the degradation exceeding 10% within the 28-day period of the test.

14C-Bronopol can therefore be evaluated as not readily biodegradable in this carbon dioxide evolution test, based on the quantitative determination of the formed carbon dioxide in the test substance assays by comparison with the calculated maximal theoretical carbon dioxide production.

Based on the rate of biodegradation of 20% CO2/ThCO2 at the end of exposure, the test substance 14C-Bronopol can be further evaluated as moderately or partly biodegradable in this test.

Descriptive statistics (mean, standard deviation) were used where applicable. The degree of biodegradation was calculated as mean of the values from replicates at the end of exposure.

Recoveries at the end of exposure are calculated as the respective mass balance (in % of total applied radioactivity) by summing up the 14CO2, radioactivity determined in the test medium after acidification, combustion of sludge, wash fractions from test vessels and centrifuge tube as well as measurement of other inorganic volatiles and organic volatiles. The targeted recovery range was from 90% to 110%, but this is not used as criteria for acceptance of the test. In aqueous phase after centrifugation, about 51 ±1% TAR and in sludge about 2±1% TAR was found. No organic or other inorganic volatiles were detected. In wash fractions from both test vessels and centrifuge tubes, only a sum of around 0.3% TAR was recovered. Taking into account of the overall recovery of the 14C measured at end of the study, about 73 ±1% TAR % of the initial applied test concentration could be recovered as the sum from various fractions.


Low mass balance recovery at the test end can be attributed either due to a loss of very small amounts of 14CO2 during sampling process or due to the production of a volatile component that was not capturable with the used absorption traps. The relatively lower deviation between replicates however indicated that the loss of 14CO2 can be excluded. Any other analysis to determine the unidentifiable radioactivity was beyond the scope of this study.

The results in this study are consistent with all validity criteria and the test is valid according to the test guideline of this study. No deviations from the test guidelines or other incidents occurred during the course of the reported test, which may have influenced the results.

Executive summary:

Under test conditions the test item was not readily biodegradable.

 

Description of key information

Moderately biodegradable 

Additional information

The key study was conducted according to OECD 301 B using radiolabelled test item. The test item concentration was set as 30 µg/L as there was toxicitiy against the inoculum observed in higher concentrations. A degradation of 20 % was oberved after 28 days.

 

A test on ready biodegradability was performed, which followed a modified procedure based on OECD guideline 301B [BASF MicroCheck 1999]. 14C-labelled bronopol was used and in deviation from the guideline, the methods were modified with respect to the test substance concentration, incubation vessels and method of determining the test substance mineralisation. In fact, the test substance concentration of 0.1 mg/L was chosen to avoid bacteria toxicity; the yield of CO2 derived from the test substance was measured as mineralisation to 14CO2 by liquid scintillation counting (LSC) and 14C-material present in the cells was determined by means of combustion analysis using a biological oxidiser. Two experiments were undertaken.

In experiment 1, around 10 % of mineralisation was reported for day 1 wehereas 55 % mineralisation was achieved within the 10 day window and ca. 89 % after a period of 29 days. In experiment 2, around 10 % degradation appeared within the first two days, followed by 45 % degradation during the 10 -day window. After 29 days, 67 % mineralization was achieved. For the radioactivity related to cell biomass, a mean value of 12 % was found to be associated with cell biomass by material alance measurements at day 29, bringing the total biodegradation in experiment 1 to 100 % at day 29 and to about 80 % for experiment 2. Considering the results of both experiments, a mean degradation rate of about 90 % of the initial 14C derived from the test substance bronopol (0.1 mg/L) was shown to be biotransformed by day 29, and consisted of about 78 % 14 CO2 and about 12 % 14C incorporated into microbial biomass.

In both experiments, the 10 -day widow acceptance criterion for "ready biodegradability" was not achieved. However, considering the additional 12 % recovery of radioactivity in the microbial cell biomass and assuming that this percentage was converted to cell biomass by day 10 (as reported in Boots MicroCheck, ELL/834), the degree of biodegradation achieved within the 10 day window would be >= 60 % (i.e. 67 %) for experiment 1 and about 57 % for experiment 2. Therefore, bronopol can be regarded as readily biodegradable in the test system.

Supportingly, an inherent biodegradability test was conducted according to modified procedures based on OECD guidelines 304 A and 302 B [Boots MicroCheck 1994]. During this test, bronopol was completely biodegraded by microorganisms within 3 days. A major metabolite (similar to 2 -nitropropane-1,3 -diol) appeared around day 3 which was again subjected to rapid and complete biodegradation on day 17. At the end of the test (on day 64), biodegradation was quantified as 68 and 23 % related to the CO2 and biomass production, respectively.

Additionally to the screening studies, there is one simulation test available which assesses the biodegradability of bronopol discharged in wastewater. The test was conducted under GLP and according to OECD guideline 314B [BASF SE 2012]. The outcomes of this study showed that 0.5 mg/L [14C]bronopol is degraded to 99% within 1 hour in municipal activated sludge which provides further confirmation of the ready biodegradability of bronopol.