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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:
30 May -29 June 2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reaction products of (Z)-octadec-9-enol and lactic acid
IUPAC Name:
Reaction products of (Z)-octadec-9-enol and lactic acid
Test material form:
liquid
Specific details on test material used for the study:
Test substance name: Dermol OL
CAS number: 42175-36-0
Batch number: P7610
Purity: 100%
Molecular formula: C21H40O3 (Chemical book)
Molecular weight: 340.5405 (Chemical book)
Receipt date: 01 February 2018
Retest date: January 2020
Storage conditions: Room temperature (15-30ºC)

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A sample of activated sludge was collected from one of the return lines at Burley
Menston sewage treatment works (West Yorkshire, UK), which has a predominantly
domestic waste-water catchment. The sample was transported at an ambient
temperature in a closed container, but with an adequate headspace, to prevent the
sample becoming anaerobic. On arrival, the sample was aerated using a compressed
air supply.
The suspended solids concentration of the activated sludge was determined by
filtering a subsample (25 mL) through a pre-dried and pre-weighed filter. The filter
and retained solids were then dried in microwave on full power and re-weighed. The
weight of the sludge solids was determined from the difference in the weights before
and after drying. The concentration of suspended solids for the test was calculated to
be 7.76 g/L (replicate range: 7.56-8.00 g/L).
The activated sludge used in this study was not acclimatised or adapted to the test
substance prior to exposure under the test conditions.
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
3.04 other: mg/ml
Based on:
other:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test System
Activated Sludge Inoculum
A sample of activated sludge was collected from one of the return lines at Burley
Menston sewage treatment works (West Yorkshire, UK), which has a predominantly
domestic waste-water catchment. The sample was transported at an ambient
temperature in a closed container, but with an adequate headspace, to prevent the
sample becoming anaerobic. On arrival, the sample was aerated using a compressed
air supply.
The suspended solids concentration of the activated sludge was determined by
filtering a subsample (25 mL) through a pre-dried and pre-weighed filter. The filter
and retained solids were then dried in microwave on full power and re-weighed. The
weight of the sludge solids was determined from the difference in the weights before
and after drying. The concentration of suspended solids for the test was calculated to
be 7.76 g/L (replicate range: 7.56-8.00 g/L).
The activated sludge used in this study was not acclimatised or adapted to the test
substance prior to exposure under the test conditions.
Preparation of Test Medium
The test was conducted in an aqueous, synthetic, mineral salts medium. A test
medium concentrate was prepared in reverse osmosis (RO) 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 (KH2PO4, 8.50 g, VWR); dipotassium hydrogen
phosphate (K2HPO4, 21.75 g, VWR); disodium hydrogen phosphate dihydrate
(Na2HPO4.2H2O, 33.40 g, Fisher); ammonium chloride (NH4Cl, 0.50 g, Fisher), all
dissolved in and made up to 1 L with RO water.
(b) calcium chloride dihydrate (CaCl.2H2O, 36.40 g, VWR), dissolved in and made
up to 1 L with RO water.
(c) magnesium sulphate heptahydrate (MgSO4.7H2O, 22.50 g, Sigma-Aldrich),
dissolved in and made up to 1 L with RO water.
(d) ferric chloride hexahydrate (FeCl3.6H2O, 0.0625 g, Sigma-Aldrich) and
concentrated hydrochloric acid (HCl, 1 drop), dissolved in and made up to 250 mL
with RO water to prepare 0.25 g/L solution.
Inorganic Carbon (IC) Measurement
The IC content of the test substance suspension in mineral medium was not measured
at the beginning of the test (as per the protocol) because the test substance was known
to be insoluble. Taking samples could have removed significant amounts of test


Study Number 3202167
Final Report

Page 13 of 29

substance and invalidated the test. This was not considered to have an impact on the
validity of the test, as information provided by the sponsor suggests that the majority
of the carbon content of this compound is organic.
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 carbon dioxide (CO2) by acidification with hydrochloric acid. The CO2
was then passed to a non-dispersive infra-red (NDIR) detector. The concentration of
CO2 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 quadruplicate.
Day -1 Procedures
On the day prior to the start of the test, the mineral medium (7 L) concentrate was
inoculated with activated sludge (81 mL). Aliquots (500 mL) of the inoculated
mineral medium concentrate were then removed and combined with 950 mL of
ultrapure water in each test vessel. The vessels were then purged with CO2 free air
overnight to remove any residual CO2 dissolved.
Preparation of Test Vessels
The study consisted of five treatment groups as described in the table below:
Treatment Group Vessel Contents
Blank Control mineral salts medium and inoculum only
Acetone Treated Silica
Control

mineral salts medium, inoculum and acetone
treated silica
Test Suspension mineral salts medium, inoculum and test substance
treated silica
Reference Substance
(Procedural control)

mineral salts medium, inoculum, acetone treated
silica and reference substance (sodium benzoate)
Toxicity control mineral salts medium, inoculum, reference
substance (sodium benzoate) and test substance
treated silica

The purpose of the toxicity control was to assess the biodegradation of the reference
substance in the presence of the test substance, to assess whether the test substance
exhibits an inhibitory effect upon the degradative action of the microbial inoculum.
Duplicate vessels were prepared for the test substance, reference substance, blank
control and acetone treated silica control groups. A single vessel was prepared for the
toxicity control.


Study Number 3202167
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Treatment of Test Vessels
Three portions of test substance were adsorbed on to the surface of 0.2-0.3µm silica
powder and one portion was added to each test substance or toxicity control vessel.
Blank acetone treated silica portions were added to each silica control and reference
vessel.
The test substance treated silica was prepared by dissolving weighed sub-samples
(30.57 to 30.67 mg) of Dermol OL in acetone (ca 10 mL). The 3 test substance
solvent stocks were transferred to individual round bottom flasks and combined with
silica. The contents of each flask was then mixed thoroughly and the acetone was
removed by rotary evaporation to leave dry test substance coated silica. The test
substance coated silica was removed from the flask into a temporary vessel. A rinse of
the test substance vessel and round bottom flask was then performed using a second
batch of untreated silica (ca 0.3 g) and acetone (ca 10 mL). The contents of the flask
were thoroughly mixed (ensuring the entire surface area of the flask exposed to the
acetone) and then the acetone was removed. The coated silica was combined with the
initial portion and one further rinse was performed as before, with a third batch of
untreated silica (ca 0.3 g) and acetone (ca 15 mL). Once the solvent was removed
from the third batch of silica, the two initial batches were returned to the round
bottom flask and then the flask was placed on a freeze drier overnight to remove any
residual acetone.
The acetone treated silica was prepared by adding silica (ca 0.9 g) and acetone
(20 mL) to a round bottom flask. The contents of the flask were then mixed and the
acetone removed by rotary evaporation until the silica was dry. The flask was then
placed on a freeze drier overnight to remove any residual acetone.
Day 0 - Test Initiation
Following all test and reference substance treatments and addition of the inoculated
medium concentrate, the volume in each vessel (including the blank control vessels)
was made up to 1.5 L by addition of appropriate amounts of UHP water. 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.
Incubation Conditions
The test vessels were incubated in the dark under the conditions below.
Temperature
The incubation and test measurements were conducted at a target temperature range
of 22 ± 2°C.
Measurement of pH
Measurements of pH were made in all vessels on day -1, the blank control, silica
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.

a The test substance was added by direct addition using silica. pH measurements were not, therefore, performed in
the test and toxicity control vessels on Day 0 to prevent possible losses of the test substance.


Study Number 3202167
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Air Flow
The air used in this study was delivered from a cylinder of CO2-free air (Air Products)
and the flow was regulated in two stages. Initial control was provided by a gas
regulator and the air flow to each vessel was controlled by individual needle valves.
Measurements of the flow rate exiting each test vessel were made at weekly intervals,
with a bubble flow meter and stopwatch. Adjustments were made as necessary to
maintain a flow rate of ca 50 mL per minute. During the test, the flow rates were
maintained within the flow rate range of 35 to 60 mL/minutes.
Sample Analysis
At appropriate intervals, the air supply to each vessel was interrupted by the trap
nearest to the test vessel being 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 each series of trap bottles was connected to the test vessel, the
air supply restarted. The initial barium hydroxide stock concentration and the residual
concentrations in detached trap bottles were determined by titration against
hydrochloric acid (nominally 0.05 M) 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 analysis of the trap solutions 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
to drive off any remaining carbon dioxide in the test and control solutions. Final
sampling and titrations were carried out on Day 29, when all of the traps in each
series were sampled.
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.
௔௕௦ × ܲ௖ × 3.667ܦ = ଶܱܥܶ
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 and/or reference substance
3.667 = the weight (mg) of CO2 produced from 1 mg of carbon.


Study Number 3202167
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The theoretical CO2 yield for the quantity of Dermol OL applied to the test vessels
and toxicity control vessel was 83 mg CO2. Theoretical yields for the quantities of
applied sodium benzoate in the toxicity control vessel and reference vessels were also
83 mg CO2. The combined theoretical yield from the toxicity control vessel was
therefore 166 mg CO2. The purpose of this toxicity 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 83 mg CO2 expected from
the sodium benzoate alone and was corrected for the test substance degradation.
Measured CO2 Yields as a Percentage of Theoretical
Biodegradation (Dt) of the reference substance and of Dermol OL expressed in terms
of percentage theoretical CO2 yield was calculated by applying the formula:

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.
Validity Criteria
The study results were considered valid if the following criteria were met:
 the IC content of the test substance suspension in mineral medium was less
than 5% of the total carbon content at the beginning of the testb
 degradation of the reference material reached 60% by Day 14
 duplicate percentage biodegradation values for vessels containing the test
substance differed from one another by less than 20%; and
 total CO2 production from each blank control vessel did not exceed 70 mg/L
(considered to be the guideline limit)
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
60
Sampling time:
10 d
Details on results:
With the exception of the inorganic carbon (IC) measurement, all the validity criteria
for the test were satisfied (degradation of reference material, CO2 production from
blank controls). The IC content of the test substance could not be measured due to the
low test substance solubility.
No inhibitory effect was observed on the biodegradation of the reference substance in
the presence of Dermol OL.
The mean biodegradation of the test substance, Dermol OL, was > 60% at the end of
the 10 day window (ca Day 11) and therefore, the test substance was considered to be
ready biodegradable under the conditions of this test.

Any other information on results incl. tables

Inorganic Carbon (IC) Content of the Test Medium

It was not possible to determine the IC content of the test substance suspension in

mineral medium at the beginning of the test, as the test substance was not sufficiently

soluble to conduct this measurement. This was not considered to have an impact on

the validity of the test, as the test substance itself was predominately organic carbon

and very pure, so it was unlikely that there was ≥ 5% inorganic carbon present.

As it was not possible to measure the IC content of the test substance suspension, the

IC content of the concentrated mineral medium was measured instead to determine

the background IC levels in each vessel. The IC content of the mineral medium was

determined to be 0.79 mg Carbon/L, which was equivalent to 1.73% of the total

carbon added to each test vessel.

Measurement of pH

The pH measurements recorded during the test are presented in Table 1.

Measured CO2 Yields as a Percentage of Theoretical CO2 Production

Carbon dioxide evolution and percentage biodegradation data are presented in Table 2,

Table 3 and Figure 1.

The mean total carbon dioxide production in the blank control and silica control

vessels was 30.11 and 31.05 mg/L, respectively, at the end of the test (Day 28 (post

acidification) and therefore, the validity criterion of less than 70 mg/L was satisfied.

As the silica control vessels showed very similar total carbon dioxide production to

the blank control vessels it is accepted that the addition of silica gel did not contribute

to CO2 production and therefore showed no biodegradation.

Test Substance

To be considered “ready biodegradable”, a test substance must achieve 60%

biodegradation by the end of the test. Additionally, the test substance must biodegrade

by at least 60% within 10 days of having reached a degradation level of 10%.

The 10 day window for the test substance vessels began (10% biodegradation level

achieved) between Days 1 and 4 (estimated at Day 2). The mean biodegradation in the

test substance vessels on Day 11 was 65% (Replicate 7: 60%; Replicate 8: 69%). As a

biodegradation level of ≥ 60% was achieved within 10 days of the 10% level being

reached, the test substance can be classified as readily biodegradable.

The percentage biodegradation values for replicate 7 and 8 at the end of the 10 day

window (Replicate 7: 60%; Replicate 8: 69%) and the end of the test (Replicate 7:

72%; Replicate 8: 87%) remained within 20% (maximum: 15%) and therefore, the

guideline validity criteria (difference < 20%) was achieved for the test substance.

Sodium Benzoate

Rapid carbon dioxide generation commenced immediately in the procedural control

vessel and declined to a more gradual rate over the period of the incubation as shown

in Figure 1.

The mean percentage biodegradation of the reference substance exceeded 60% by

Day 6 (replicate 5: 57%; replicate 6: 63%) and had reached a mean biodegradation of

84% (replicate 5: 80%; replicate 6: 89%) by the end of the test (Day 28 - post

acidification). The validity criterion of 60% biodegradation of the reference substance

by 14 days was therefore met.

Toxicity Control

Total biodegradation in the toxicity control based on both sodium benzoate and

Dermol OL was found to be 84% at the end of the test, 60% was reached by day 11

(64%), which is greater than the required 25%.

Assessment of biodegradation in the toxicity control when confined to the sodium

benzoate fraction only and corrected for Dermol OL achieved the reference substance

pass of 60% by Day 11 (63%) and 88% by the end of the test.

The percentage biodegradation of the reference substance in the presence of

Dermol OL indicated that there were no inhibitory effects from the test substance on

the sludge microorganisms over the full duration of the test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
With the exception of the inorganic carbon (IC) measurement, all the validity criteria
for the test were satisfied (degradation of reference material, CO2 production from
blank controls). The IC content of the test substance could not be measured due to the
low test substance solubility.
No inhibitory effect was observed on the biodegradation of the reference substance in
the presence of Dermol OL.
The mean biodegradation of the test substance, Dermol OL, was > 60% at the end of
the 10 day window (ca Day 11) and therefore, the test substance was considered to be
ready biodegradable under the conditions of this test.
Executive summary:

The ready biodegradability of the test substance Dermol OL was assessed by

measurement of carbon dioxide (CO2) evolution under standard conditions over

28 days. The study was conducted in accordance to the OECD Guideline 301B,

Ready Biodegradability (Adopted 1981, Revised 1992).

The test substance was added to the test system on silica. Silica was used for test

substance addition in the reported test because this was considered to be the most

appropriate application method due to the low solubility.

Buffered mineral salts medium was added to give a test substance concentration

equivalent to 15 mg carbon/L. The medium was inoculated with microorganisms

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 removed from the series and

their contents titrated against 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 remaining traps on Day 29 was performed following

overnight aeration.

The performance of the inoculum was 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 and blank silica control vessels were also

prepared. The results of the blank silica control vessels were used to check the

validity of the test and to correct for baseline CO2 evolved.

The mean biodegradation in the test substance vessels met the ready biodegradation

pass level (60%) with 65% biodegradation on Day 11 (replicate 7: 60%;

replicate 8: 69%). The pass level was achieved within a 10 day window, as the 10%

level was estimated to have been achieved at day 2 (percentage biodegradation at day

1, replicate 7: 4%; replicate 8: 3% and percentage biodegradation at day 4,

replicate 7: 27%; replicate 8: 28%).

The mean total CO2 production in the blank control and blank silica control vessels

was 30.11 and 31.05 mg/L at the end of the test, satisfying the validity criterion of

< 70 mg/L.

The inoculum was considered appropriate for the test, as the mean biodegradation of

the reference substance vessels reached 60% by Day 6 (replicate 5: 57%;

replicate 6: 63%) and had reached a mean biodegradation of 84% (replicate 5: 80%;

replicate 6: 89%) by the end of the incubation (Day 28 - post acidification).

The percentage biodegradation of the reference substance in the presence of

Dermol OL (toxicity control) achieved the reference substance pass (60% when

calculated on reference only and corrected for test substance degradation) by Day 11

(63%). The biodegradation for the toxicity control calculated for combined test and

reference substance achieved 84% degradation at Day 29, satisfying the guideline

required 25% degradation by the end of the test period. These results indicate that

there were no inhibitory effects from the test substance on the sludge microorganisms

over the full duration of the test.

It was not possible to determine the IC content of the test substance suspension in

mineral medium at the beginning of the test, as the test substance was not sufficiently

soluble to conduct this measurement. This was not considered to have an impact on

the validity of the test, as information provided by the sponsor suggests that the

majority of the carbon content of this compound is organic.

As it was not possible to measure the IC content of the test substance suspension, the

IC content of the concentrated mineral medium was measured instead to determine

the background IC levels in each vessel. The IC content of the mineral medium was

determined to be 0.79 mg Carbon/L, which was equivalent to 1.73% of the total

carbon added to each test vessel.

All validity criteria except the IC content (which was not measured as per the protocol)

were satisfied and therefore the test was considered to be valid.

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