Nitrile hydratase recombinant from E.coli

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:

31 December 2020 to 08 March 2021

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)

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:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 01 February 2021 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection.

Duration of test (contact time):

28 d

Initial conc.:

119.6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines. The deionised reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).
- Test temperature: The test was carried out in a temperature controlled room at temperatures of between approximately 20 and 23 °C.
- pH: 7.5 (day 0); 7.3-7.5 (day 28)
- pH adjusted: no
- Suspended solids concentration: The suspended solids concentration was equal to 1.4 g/L prior to use. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5L test culture vessels each containing 3 L of preparation
- Number of culture flasks/concentration:

1) An inoculated control, in duplicate, consisting of inoculated mineral medium
2) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L
3) The test material, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L
4) The test material plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only)

- Method used to create aerobic conditions: Approximately 24 hours prior to addition of the test and reference materials the vessels were filled with 2400 mL of mineral medium and 64.3 mL of inoculum and aerated overnight. The volume in all of the vessels was adjusted to 3 L by the addition of mineral medium, which had been purged overnight with CO2-free air. The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a moisture trap prior to being passed through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using deionised reverse osmosis water.
- Other: In order to confirm that the sodium benzoate solution was prepared correctly, a diluted 100 mg/L stock solution (in reverse osmosis water) was sampled for TOC analysis.
- Test material preparation: As the test material was a suspension and stored frozen, it was defrosted in a water bath at 25 °C for 55 minutes and shaken several times prior to weighing out. An amount of test material (358.8 mg) was dispersed directly in the inoculated mineral medium. The volume was adjusted to 3 L to give a final concentration of 119.6 mg/L, equivalent to 10 mg carbon/L. A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

SAMPLING - INORGANIC CARBON ANALYSIS
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. All samples were analyzed for Inorganic Carbon (IC) immediately. The remainder of all samples (with the exception of the Day 0 samples which were discarded) were frozen for further analysis if required. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
- Sampling method: The samples were analysed for IC using either a Shimadzu TOC-VCSH TOC analyser or a Shimadzu TOC-LCSH TOC analyser. Samples (50 or 135 μL) were injected into the IC channel of the TOC analyser. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in at least triplicate with three replicates being used in the calculation.

SAMPLING - INORGANIC CARBON/TOTAL CARBON RATIO
- Sampling frequency: Samples (30 mL) were removed from the test material vessels on Day 0 prior to the addition of the test material in order to calculate the IC content in the test media. The samples were filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL used to pre-condition the filter was discarded) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL used to pre-condition the filter was discarded) prior to DOC analysis.
- Sampling method: Inorganic Carbon/Total Carbon analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water. The samples were analysed for IC and Total Carbon (TC) using a Shimadzu TOC-VCPH TOC Analyser. Samples (50 μL) were injected into the TC and IC channels of the TOC analyzer. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in at least triplicate with three replicates being used in the calculation.

CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculated mineral medium
- Toxicity control: A toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test. An amount of test material (358.8 mg) was dispersed directly in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 L to give a final concentration of 119.6 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

ASSESSMENTS
- Observations: The appearance of the test preparations was recorded on Days 0, 7, 14, 21 and 28.
- pH measurements: The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ30d Flexi handheld meter.

DATA EVALUATION
- Determination of carbon content: The test material contains 8.36 % carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/L, which was equivalent to 119.6 mg test material/L, the total organic carbon present was 30 mg C in 3 L.

The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated as follows:

No of C atoms x mol wt of C / mol wt of sodium benzoate x 100
7x12.011 / 144.11 x 100 = 58.34%

Thus for a 10 mg C/L test concentration, which was equivalent to 17.1 mg reference item/L, the total organic carbon present for sodium benzoate was 30 mg C in 3 L.

- Percentage biodegradation: The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the IC values into the following equation. The values of Replicates 1 and 2 are meaned for the inoculum control, test and reference materials before substitution into the following equation (The conversion factor for carbon to carbon dioxide is 3.67):

% ThCO2(=%biodegradation∗)= mg IC in test flask−mg IC in control flask / mg TOC added as test chemical x100

- Total CO2 evolution: The total CO2 evolution in the inoculum control vessels on Day 28 of the test is calculated from the equation below. The IC values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:

Total CO2 evolution (mg/L) =mg IC in control x (100 / %C of CO2) x 1/test volume

= =mg IC in control x (100 / 27.29) x 13

VALIDATION CRITERIA
The results of the degradation test are considered valid if in the same test the reference item yields ≥60% degradation (in a 10-day window) by Day 14.
The test material may be considered to be readily biodegradable if ≥60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10 %.
The toxicity control (test material and sodium benzoate) should attain ≥25% degradation by Day 14 for the test material to be considered as non-inhibitory.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the time the plateau is reached, at the end of the test or at the end of the 10-day window, as appropriate, is less than 20 %.
The total CO2 evolution in the inoculum control vessels at the end of the test should not normally exceed 40 mg/L medium (= 120 mg/3 L, corresponding to 33 mg C per flask); however values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.
The IC content of the test material suspension in the mineral medium at the beginning of the test should be <5 % of the TC.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

97

Sampling time:

28 d

Details on results:

The total CO2 evolution in the inoculum control vessels on Day 28 was 39.14 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test material suspension in the mineral medium at the start of the test, before dosing, was below 5 % of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20 % and hence satisfied the validation criterion given in the OECD Test Guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels.

The IC analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test material attained 97 % biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Results with reference substance:

The toxicity control attained 56 % biodegradation after 14 days and 66 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 75 % biodegradation after 14 days with greater than 60 % degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines. After 28 days 89 % biodegradation was attained.

Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test Material	Toxicity Control
0	0	0	0
2	51	28	29
6	63	56	50
8	68	59	51
10	73	65	53
14	75	79	56
21	77	83	59
28	83	98	62
29*	89	97	66

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of this study, the test material attained 97 % biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and OSCPP 835.3110, under GLP conditions. 

The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between approximately 20 and 23 °C for 28 days.
The biodegradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Sodium benzoate attained 75 % biodegradation after 14 days with greater than 60 % degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Under the conditions of this study, the test material attained 97 % biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

Under the conditions of this study, the test material attained 97 % biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test material was assessed in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and OSCPP 835.3110, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). 

The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between approximately 20 and 23 °C for 28 days.
The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Sodium benzoate attained 75 % biodegradation after 14 days with greater than 60 % degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Under the conditions of this study, the test material attained 97 % biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.