Registration Dossier

Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

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:
01 February 2016 to 23 June 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The carbon content, 68.2 %, was calculated from the molecular weights and formulae provided by the Sponsor. This carbon content was used in setting the test concentration of the test material. No allowance was made for purity.
A solubility trial was performed by weighing a sub-sample (33.04 mg) of test material and attempting to dissolve this in laboratory water (1.5 L). The mixture was stirred for 24 hours but the test substance was still visible.
An adsorption to silica trial was performed using 3 g and then 0.1 g portions of silica before the following method was selected.
The test substance was adsorbed on to the surface of silica powder which was dispersed through the samples. A weighed sub-sample (65.97, 65.98 or 66.00 mg) of the test substance was placed in a round bottomed flask along with silica (ca 0.3 g) and acetone (ca 5 mL). The contents were mixed thoroughly and then the solvent was evaporated off. This then left the silica with a coating of the test material remaining on it. The test material coated silica was removed. Additional untreated silica (ca 0.3 g) was added to the round bottomed flask and untreated acetone (ca 5 mL) was added to it. The acetone was evaporated off and the coated silica portions combined. A third portion of untreated silica (ca 0.3 g) was added to the round bottomed flask and untreated acetone (ca 5 mL) again added. This further addition of silica and solvent was followed by evaporation and the coated silica again removed and combined with the earlier portions to ensure no test material remained in the flask. Samples were then subjected to freeze drying overnight. The test substance was added directly to the test vessels in this form.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Storage conditions: The sample was transported in a closed container, but with an adequate headspace, to prevent the sample becoming anaerobic. On arrival, the sample was aerated by means of a compressed air supply.
- Pretreatment: The activated sludge used in this study was not deliberately acclimatised or adapted to the test material before exposure under test conditions.
- Initial cell/biomass concentration: The suspended solids concentration of the activated sludge was determined by filtering a subsample (25 mL) through a pre-dried and pre-weighed glass microfibre filter (Whatman GF/C). The filter and retained solids were then dried in an oven (nominally 105 °C) and re-weighed. The weight of the sludge solids was determined from the difference in the dry weights before and after filtering. The concentration of suspended solids was calculated to be 7.21 g/L.
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
other: organic carbon/L.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The test was conducted in an aqueous, synthetic, mineral salts medium. A test medium concentrate was prepared in ultra-pure water containing 30 mL/L solution (a) and 3 mL/L of each of solutions (b), (c) and (d). Solutions (a) to (d) were prepared as follows:

(a) potassium dihydrogen phosphate (8.50 g, VWR, ≥ 99.0 %); dipotassium hydrogen phosphate (21.75 g, Fisher, ≥ 99.0 %); disodium hydrogen phosphate dihydrate (33.40 g, Sigma-Aldrich, ≥ 99.0 %); ammonium chloride (0.50 g, Fisher, >99 %), all dissolved in and made up to 1 L with RO water.

(b) calcium chloride dihydrate (36.40 g, VWR, Analar Normapur), dissolved in and made up to 1 L with RO water.

(c) magnesium sulphate heptahydrate (22.50 g, Sigma-Aldrich, ≥ 99.0%), dissolved in and made up to 1 L with RO water.

(d) ferric chloride hexahydrate (0.25 g, Sigma-Aldrich, 97 %) and concentrated hydrochloric acid (1 drop, VWR, Analar Normapur), dissolved in and made up to 1 L with RO water.

On the basis of the suspended solids determination described previously, the medium was inoculated with activated sludge (100 mL in a total volume of 8 L) to give a suspended solids concentration of 90 mg/L. This provided a nominal final solids concentration of 30 mg/L in each test vessel (1 L added to a total volume of 3 L).
The inorganic carbon (IC) concentration of the inoculated mineral salts medium was determined using an InnovOx carbon analyser. In this analysis, IC in the samples was released as CO2 by acidification with hydrochloric acid. The CO2 was then passed to a non-dispersive infra red (NDIR) detector. The concentration of carbon dioxide was determined in the NDIR detector, by measuring the amount of infra-red energy absorbed by the sample. A calibration check was performed on each occasion by injecting a series of sodium hydrogen carbonate standards. The existing calibration curve was used to quantify the IC present in the samples. Each sample was analysed in triplicate.

- Test temperature: The incubation and test measurements were conducted at a nominal temperature range of 22 ± 2 °C.
- pH: Measurements of pH were made in the blank control and reference substance vessels at the start of incubationa and in all vessels at the end of the test prior to the addition of the hydrochloric acid. Measured pH values ranged from pH 7.38 to pH 7.50 on Day 0 and pH 7.22 to pH 7.41 on Day 28.
- Continuous darkness: Yes. The test vessels were incubated in the dark.

TEST SYSTEM
- Treatment of vessels: The test material was accurately weighed (65.97 to 66.00 mg) for addition to test substance and toxicity control vessels as a silica coating, to give a nominal test substance concentration corresponding to 15 mg carbon/L.
Silica, treated with acetone but no test substance was added to blank control and reference vessels.
- Number of culture flasks/concentration: Duplicate vessels were prepared for the test substance, reference substance and blank control groups. A single vessel was prepared for the toxicity control.
The volume in each vessel (including the blank control vessels) was made up to 3 L by addition of ultra-pure water.
- Method used to create aerobic conditions: The air used in this study was delivered from a cylinder of CO2-free air (Air Products) and was regulated in two stages. Initial control was provided by a gas regulator and the air flow to each vessel controlled by individual needle valves. Measurements of the flow rate exiting each test vessel were made at intervals not exceeding seven days, with a bubble flow meter and stopwatch. Adjustments were made as necessary to maintain a flow rate of ca 50 mL per minute.
- Test performed in closed vessels: Yes. Each vessel was sealed, connected to a series of three traps containing aqueous barium hydroxide (nominally 0.0125 M), and the carbon dioxide-free air supply initiated.
- Details of trap for CO2 and volatile organics: In this analysis, IC in the samples was released as CO2 by acidification with hydrochloric acid. The CO2 was then passed to a non-dispersive infra-red (NDIR) detector. The concentration of carbon dioxide was determined in the NDIR detector, by measuring the amount of infra-red energy absorbed by the sample. A calibration check was performed on each occasion by injecting a series of sodium hydrogen carbonate standards. The existing calibration curve was used to quantify the IC present in the samples. Each sample was analysed in triplicate.

SAMPLING
- Sampling analysis: At appropriate intervals, the air supply to each vessel was interrupted and the trap bottle nearest to the test vessel was removed for sampling. The remaining two bottles of the series were moved towards the test vessel, and a fresh trap bottle placed on the end of the series. Once the series of trap bottles were connected to the test vessel the air supply was restarted. The initial barium hydroxide stock concentration and the residual concentrations in detached trap bottles were determined by titration against hydrochloric acid (nominally 0.05M) using 0.5% ethanolic phenolphthalein indicator solution. Titrations were performed on 20 mL trap solution volumes until two matching (± 0.1 mL) titres were obtained.
Evolved CO2 from the vessels was trapped in the barium hydroxide traps by formation of a barium carbonate precipitate. This resulted in a decrease in the concentration of barium hydroxide. Consequently, the amount of evolved CO2 was calculated from the decrease in the barium hydroxide concentration, determined by titration against hydrochloric acid.
Following the trap analysis on Day 28, each culture vessel was opened and concentrated hydrochloric acid (1 mL) added. The vessels were then reconnected to
the series of trap bottles and aeration continued until the following day. The acidification and aeration procedure drives off generated carbon dioxide remaining in solution. Final sampling and titrations were carried out on Day 29, when all of the traps in each series were sampled.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Vessel contained inoculated mineral salts medium.
- Toxicity control: A single vessel contained inoculated mineral salts medium, test substance and sodium benzoate. The purpose of the toxicity control was to assess the biodegradation of the reference substance in the presence of the test substance.
- A reference substance stock solution (2.25 g carbon/L) was prepared by dissolving sodium benzoate (3.86 g) in ultra-pure water (1 L). Reference and toxicity control vessels were treated with the stock solution (20 mL), to give a nominal sodium benzoate concentration corresponding to 15 mg carbon/L.
Based on the molecular formula of sodium benzoate, its carbon content expressed as a percentage by weight is 58.3 %. This value was used in setting the test concentration of sodium benzoate. Duplicate vessels were prepared for the reference substance.

CALCULATIONS
- Theoretical CO2 Yields: The theoretical yield of carbon dioxide (TCO2 in mg) from cultures containing the test and/or reference substances was calculated as shown below.

TCO2 = Dabs x Pc x 3.667

where:
Dabs = the absolute dose i.e. the amount (mg) of test or reference substance added to the culture
Pc = the percentage carbon content of the test or reference substance
3.667 = the weight (mg) of CO2 produced from 1 mg of carbon

The theoretical CO2 yield for the quantity of test material applied to the test vessels and toxicity control vessel was 165 mg CO2. Theoretical yields for the quantities of applied sodium benzoate in the toxicity control vessel and reference vessels were also 165 mg CO2. The combined theoretical yield from the toxicity control vessel was 330 mg CO2. The purpose of this control was not to assess the extent of degradation of the entire mixture, but instead to assess the impact of the presence of the test substance on the degradation of the reference material. The theoretical yield in this case was, therefore, limited to the 165 mg CO2 expected from the sodium benzoate alone and the actual yield corrected for that from the test substance.

- Measured CO2 Yields as a Percentage of Theoretical

Biodegradation (Dt) of the reference substance and of test material expressed in terms of percentage theoretical CO2 yield was calculated by applying the formula:

Dt = (cumulative mg CO2 produced at a time (t) / 165) x 100

The cumulative CO2 values for the test substance, reference substance and toxicity control were corrected for the mean CO2 generated by the blank controls, to determine the percent biodegradation.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
INORGANIC CARBON (IC) CONTENT OF THE TEST MEDIUM
The inorganic carbon content of the concentrated test medium was determined to be 2.59 mg carbon/L which is more than 5 % (2.25 mg/L) of the total carbon content at the beginning of the test (15 mg/L). However, there is no evidence of suppression of biodegradation from the background carbon as the reference substance vessels produced carbon dioxide as expected.
Additionally, the conclusion that the test substance is not readily biodegradable is substantiated by the low level of carbon dioxide produced by the test substance vessels. The carbon dioxide determined in these traps was far less than the required amount needed to meet the ready biodegradable criteria even without the background subtraction of the blank controls.
In summary, considering the higher than normal background inorganic carbon would have been consistent in all vessels and accounted for in the calculations using the values from the blank controls, the results and conclusions of the study are valid.

MEASURED CO2 YIELDS AS A PERCENTAGE OF THEORETICAL
The mean total CO2 production in the blank control vessels was 214.8 mg (71.6 mg/L) at the end of the test (Day 28). Although slightly more than the
validity criterion of < 70 mg/L, the excess can be explained by degradation of residual acetone from the silica coating process which will have been common to all samples.
Any effect of this excess will have been cancelled by subtraction of control results from all samples including reference samples which showed normal degradation.

TEST MATERIAL
Carbon dioxide evolution in the test samples minus evolution in the blank samples was used to calculate biodegradation of the test substance as a percentage of the theoretical maximum. The test material showed no biodegradation during the study with mean % biodegradation not exceeding 0 %. The test material
cannot, therefore, be considered readily biodegradable.
Percent biodegradation values at each sampling interval, for the two replicates containing the test material with propane-1,2-diol did not vary, therefore satisfying the validity criterion of less than 20 % difference.
Results with reference substance:
Rapid carbon dioxide generation commenced immediately and declined to a more gradual rate over the period of the incubation. The mean percentage biodegradation had exceeded 60 % by Day 6 (69 %) and by the end of thestudy the mean biodegradation had reached a maximum mean of 94 %. The validity criterion of 60 % biodegradation at 14 days was therefore met. Values of greater than 100 % are most likely due to a difference in the rate of carbon dioxide production in the blank control vessels when compared to the reference vessels.

Validity

All validity criteria were satisfied excluding the inorganic carbon content of the test medium to be less than 5 % of the total carbon and the production of CO2 from control samples, which were marginal, common to all samples, accounted for in calculation of results and which did not, therefore, affect the outcome of the study.

The rate of biodegradation of the reference substance in the presence of the test substance was reduced, indicating that the test material had an effect on the sludge microorganisms under the test conditions. The test was, however, considered to be valid as degradation was > 25 %.

Mean carbon dioxide evolution from the test material was 0 % of the theoretical carbon dioxide yield throughout the test. The level of biodegradation did not meet the requirements for ready biodegradability and the test material cannot, therefore, be classified as readily biodegradable.

Toxicity Control

Assessment of biodegradation in the toxicity control was confined to the sodium benzoate fraction. The rate of biodegradation of the reference substance in the presence of the test materiall (41 % at Day 6, 38 % by the end of the test) was almost half of the reference substance alone.

This suggests that the test material had an effect on the sludge microorganisms under the test conditions, however, the test was considered to be valid as degradation was > 25 % by Day 14.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of the study, the test material was determined to be not readily biodegradable.
Executive summary:

A study was conducted to evaluate the ready biodegradability of the test material in accordance with the standardised guideline OECD 301B under GLP conditions.

The ready biodegradability of the test material was assessed by measurement of carbon dioxide (CO2) evolution under standard conditions. The test substance was added to the test system as a solid adsorbed to silica dispersed through the test system. Buffered mineral salts medium was added to give a test substance concentration equivalent to 15 mg organic carbon/L. The medium was inoculated with microorganisms (30 mg/L) derived from a sample of activated sludge not previously intentionally exposed to the test substance. Test vessels were incubated in darkness at 22 ± 2 °C for 28 days and their contents continuously sparged with a supply of CO2-free air. The exhaust air from each vessel was passed through a series of traps containing a barium hydroxide solution, to trap evolved CO2.

At regular intervals during the incubation, traps were detached and their contents titrated with hydrochloric acid to determine the quantity of CO2 evolved from the respective test vessels. At the end of incubation, 28 days, the test vessel contents were acidified to release any residual CO2 that may have remained in solution. Titration of the traps was performed following overnight aeration.

The procedure and the activity of the inoculum were checked by measuring the CO2 evolved from vessels containing a reference substance, sodium benzoate. An additional vessel containing a combination of the test and reference substances served as a toxicity control to assess whether the test substance was inhibitory to biodegradation at the test concentration. Two blank control vessels were also prepared containing inoculated medium only. The results of these vessels were used to check the validity of the test and to correct the evolved CO2 values.

The test material showed 0 % biodegradation at the end of the incubation and acidification. As a result, the test material cannot be considered readily biodegradable.

Mean total CO2 production in the blank control vessels was 214.8 mg (71.6 mg/L) at the end of the test (Day 28). Although slightly more than the validity criterion of < 70 mg/L, the excess can be explained by degradation of residual acetone from the silica coating process which will have been common to all samples. Any effect will have been cancelled by subtraction of control results from all samples including reference samples which showed normal degradation. The absence of any observed degradation of test substance alongside normal reference degradation confirms the validity of the result.

Mean biodegradation of the reference substance had exceeded 60 % by Day 14 and had reached a maximum of 94 % by the end of the incubation (Day 28). The rate of biodegradation of the reference substance in the presence of the test material (41 % at Day 6, 38 % by the end of the test) suggests that the test material had a buffering effect on the sludge microorganisms under the test conditions.

Biodegradation of the reference substance in the presence of the test substance was > 25 % by Day 14 and therefore met the validity requirement.

All validity criteria were satisfied excluding the inorganic carbon content of the test medium to be less than 5 % of the total carbon and the production of CO2 from control samples, which were marginal, common to all samples, accounted for in calculation of results and which did not, therefore, affect the outcome of the study.

Under the conditions of the study, the test material was determined to be not readily biodegradable.

Description of key information

Under the conditions of the study, the test material was determined to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A study was conducted to evaluate the ready biodegradability of the test material in accordance with the standardised guideline OECD 301B under GLP conditions. The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The ready biodegradability of the test material was assessed by measurement of carbon dioxide (CO2) evolution under standard conditions. The test substance was added to the test system as a solid adsorbed to silica dispersed through the test system. Buffered mineral salts medium was added to give a test substance concentration equivalent to 15 mg organic carbon/L. The medium was inoculated with microorganisms (30 mg/L) derived from a sample of activated sludge not previously intentionally exposed to the test substance. Test vessels were incubated in darkness at 22 ± 2 °C for 28 days and their contents continuously sparged with a supply of CO2-free air. The exhaust air from each vessel was passed through a series of traps containing a barium hydroxide solution, to trap evolved CO2.

At regular intervals during the incubation, traps were detached and their contents titrated with hydrochloric acid to determine the quantity of CO2 evolved from the respective test vessels. At the end of incubation, 28 days, the test vessel contents were acidified to release any residual CO2 that may have remained in solution. Titration of the traps was performed following overnight aeration.

The procedure and the activity of the inoculum were checked by measuring the CO2 evolved from vessels containing a reference substance, sodium benzoate. An additional vessel containing a combination of the test and reference substances served as a toxicity control to assess whether the test substance was inhibitory to biodegradation at the test concentration. Two blank control vessels were also prepared containing inoculated medium only. The results of these vessels were used to check the validity of the test and to correct the evolved CO2 values.

The test material showed 0 % biodegradation at the end of the incubation and acidification. As a result, the test material cannot be considered readily biodegradable.

Mean total CO2 production in the blank control vessels was 214.8 mg (71.6 mg/L) at the end of the test (Day 28). Although slightly more than the validity criterion of < 70 mg/L, the excess can be explained by degradation of residual acetone from the silica coating process which will have been common to all samples. Any effect will have been cancelled by subtraction of control results from all samples including reference samples which showed normal degradation. The absence of any observed degradation of test substance alongside normal reference degradation confirms the validity of the result.

Mean biodegradation of the reference substance had exceeded 60 % by Day 6 and had reached a maximum of 94 % by the end of the incubation (Day 28).

Biodegradation of the reference substance in the presence of the test substance was > 25 % by Day 14 and therefore met the validity requirement.

All validity criteria were satisfied excluding the inorganic carbon content of the test medium to be less than 5 % of the total carbon and the production of CO2 from control samples, which were marginal, common to all samples, accounted for in calculation of results and which did not, therefore, affect the outcome of the study.

Under the conditions of the study, the test material was determined to be not readily biodegradable.