Ethyl nicotinate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl nicotinate

EC Number:

210-370-7

EC Name:

Ethyl nicotinate

Cas Number:

614-18-6

Molecular formula:

C8H9NO2

IUPAC Name:

ethyl nicotinate

Test material form:

liquid

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

e activated sludge used for this study was obtained from the Wareham Wastewater Treatment
Plant, Wareham, Massachusetts, which receives primarily domestic waste. Approximately 4 L
of activated sludge was collected on 24 July 2017 and transported to Smithers Viscient. Upon
arrival at Smithers Viscient, the sludge was passed through a 2-mm sieve and centrifuged at
1000 rpm for 10 minutes. The supernatant was discarded, the sludge was washed with mineral
medium (SMV No. 24July17L37-E) and the contents were centrifuged again, and the supernatant
was discarded. The moisture content of the activated sludge was determined, using a Sartorius
MA-150 automated moisture analyzer, to be 95.94% and the percent solids was determined to be
4.06%. An inoculum solution with 15 mg suspended solids/mL was prepared (73.90 g wet
weight sludge brought to 200 mL with mineral medium), stirred with a Teflon magnetic stir bar
at 22 ± 2 °C, and aerated until used. The test suspension flasks, the blank flasks, the procedural
control flask, the toxicity control flask, and the pH/TIC flask all received 6.0 mL of the inoculum
to produce an activated sludge concentration of 30 mg solids/L.

In addition, a 50-g aliquot of fresh soil (Station Street, Wareham, MA), a 25-g aliquot of
Taunton River sediment, and a 25-g aliquot of Weweantic River sediment were collected near
Smithers Viscient. The soil and sediments were suspended in 1 L of Weweantic River water by
hand shaking for one minute and the suspension was then filtered through glass wool. This
soil/sediment inoculum was determined to contain 0.2% solids. A 3.0-mL aliquot of the
soil/sediment filtrate was added to each test vessel containing mineral medium and 6.0 mL of
activated sludge Table 2.

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 10 mg/L

Based on:

ThCO2

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Calibration Standards
The inorganic calibration standard for liquid total inorganic carbon (TIC) analysis,
sodium bicarbonate (SMV No. 8233), Batch No. 0000125544, CAS No. 144-55-8, was received
from Fisher Scientific, Florence, Kentucky on 29 April 2016 with a stated purity of 100.1%
(tested as 100%).

The calibration standard, potassium hydrogen phthalate (SMV No. 8224), Lot No. M14C033,
was received from Alfa Aesar, Haverhill, Massachusetts on 27 April 2016 with a stated purity of
99.96% (tested as 100%).

Standard Reagents
All aqueous solutions were prepared using purified reagent water generated from a Millipore
water purification unit. The source water for this system is well water that has been shown to be
free of all contaminants which could affect study results. Treatment in the system further
purified the water prior to use. The 0.45-µm filtered purified reagent water typically has total
organic carbon below 1 mg/L. The dissolved organic carbon (DOC) content of the reagent grade
water was shown to be 1.1 ppm prior to use. Using a Shimadzu TOC-L/ASI-L/SSM analyzer,
DOC values were compared to a standard curve of at least five concentrations of potassium
phthalate in order to determine DOC content.

A 1 M hydrochloric acid solution was prepared by diluting 165 mL of hydrochloric acid to a
final volume of 2000 mL with purified reagent water.

A 0.2 N potassium hydroxide (KOH) solution was prepared by weighing out 52.81 g (44.89 g as
active ingredient) of KOH and diluting to a final volume of 4000 mL with purified reagent water.
All chemicals were at least reagent grade and were obtained from commercial sources.

2.3 Stock Solution Preparation
2.3.1 Test Substance
A 1 mg C/mL ethyl nicotinate stock solution was prepared by diluting 0.3377 g (0.2000 g as
carbon) to a final volume of 200 mL with purified reagent water. A 30-mL aliquot of the ethyl
nicotinate stock solution was added into both test vessels, the toxicity control vessel, and the pH
check vessel.

2.3.2 Reference Substance
A sodium benzoate stock solution of 17.15 mg/mL (10 mg C/mL) was prepared by bringing
0.3441 g (0.3434 g as active ingredient) of sodium benzoate to 20.02 mL with purified reagent
water. The sodium benzoate stock solution was prepared on the day of use (day 0). This stock
solution was used to prepare the procedural and toxicity controls.

2.3.3 Calibration Standards
A 500 mg C/L sodium bicarbonate stock solution was prepared by bringing 0.6993 g of
sodium bicarbonate (14.3% carbon based on the empirical formula of NaHCO
) to 200 mL with
purified reagent water. This solution was used to prepare calibration standards for the TIC
analysis for the TOC analyzer.

A 2000 mg C/L potassium phthalate stock solution was prepared by bringing 0.2130 g of
potassium phthalate (47.06% carbon based on the empirical formula of C
) to a final
volume of 50 mL with purified reagent water. This solution was used to prepare TOC
calibration standards for the TOC analyzer.

2.3 Stock Solution Preparation
2.3.1 Test Substance
A 1 mg C/mL ethyl nicotinate stock solution was prepared by diluting 0.3377 g (0.2000 g as
carbon) to a final volume of 200 mL with purified reagent water. A 30-mL aliquot of the ethyl
nicotinate stock solution was added into both test vessels, the toxicity control vessel, and the pH
check vessel.

2.3.2 Reference Substance
A sodium benzoate stock solution of 17.15 mg/mL (10 mg C/mL) was prepared by bringing
0.3441 g (0.3434 g as active ingredient) of sodium benzoate to 20.02 mL with purified reagent
water. The sodium benzoate stock solution was prepared on the day of use (day 0). This stock
solution was used to prepare the procedural and toxicity controls.

2.3.3 Calibration Standards
A 500 mg C/L sodium bicarbonate stock solution was prepared by bringing 0.6993 g of
sodium bicarbonate (14.3% carbon based on the empirical formula of NaHCO
) to 200 mL with
purified reagent water. This solution was used to prepare calibration standards for the TIC
analysis for the TOC analyzer.

A 2000 mg C/L potassium phthalate stock solution was prepared by bringing 0.2130 g of
potassium phthalate (47.06% carbon based on the empirical formula of C
) to a final
volume of 50 mL with purified reagent water. This solution was used to prepare TOC
calibration standards for the TOC analyzer.

2.3 Stock Solution Preparation
2.3.1 Test Substance
A 1 mg C/mL ethyl nicotinate stock solution was prepared by diluting 0.3377 g (0.2000 g as
carbon) to a final volume of 200 mL with purified reagent water. A 30-mL aliquot of the ethyl
nicotinate stock solution was added into both test vessels, the toxicity control vessel, and the pH
check vessel.

2.3.2 Reference Substance
A sodium benzoate stock solution of 17.15 mg/mL (10 mg C/mL) was prepared by bringing
0.3441 g (0.3434 g as active ingredient) of sodium benzoate to 20.02 mL with purified reagent
water. The sodium benzoate stock solution was prepared on the day of use (day 0). This stock
solution was used to prepare the procedural and toxicity controls.

2.3.3 Calibration Standards
A 500 mg C/L sodium bicarbonate stock solution was prepared by bringing 0.6993 g of
sodium bicarbonate (14.3% carbon based on the empirical formula of NaHCO
) to 200 mL with
purified reagent water. This solution was used to prepare calibration standards for the TIC
analysis for the TOC analyzer.

A 2000 mg C/L potassium phthalate stock solution was prepared by bringing 0.2130 g of
potassium phthalate (47.06% carbon based on the empirical formula of C
) to a final
volume of 50 mL with purified reagent water. This solution was used to prepare TOC
calibration standards for the TOC analyzer.

Stock Solution Preparation
Test Substance
A 1 mg C/mL ethyl nicotinate stock solution was prepared by diluting 0.3377 g (0.2000 g as
carbon) to a final volume of 200 mL with purified reagent water. A 30-mL aliquot of the ethyl
nicotinate stock solution was added into both test vessels, the toxicity control vessel, and the pH
check vessel.

Reference Substance
A sodium benzoate stock solution of 17.15 mg/mL (10 mg C/mL) was prepared by bringing
0.3441 g (0.3434 g as active ingredient) of sodium benzoate to 20.02 mL with purified reagent
water. The sodium benzoate stock solution was prepared on the day of use (day 0). This stock
solution was used to prepare the procedural and toxicity controls.

2.3.3 Calibration Standards
A 500 mg C/L sodium bicarbonate stock solution was prepared by bringing 0.6993 g of
sodium bicarbonate (14.3% carbon based on the empirical formula of NaHCO3) to 200 mL with
purified reagent water. This solution was used to prepare calibration standards for the TIC
analysis for the TOC analyzer.

A 2000 mg C/L potassium phthalate stock solution was prepared by bringing 0.2130 g of
potassium phthalate (47.06% carbon based on the empirical formula of C8H5KO4) to a final
volume of 50 mL with purified reagent water. This solution was used to prepare TOC
calibration standards for the TOC analyzer.

2.4 Mineral Medium
The aqueous medium for testing provided the essential nutrients, except for carbon, necessary to
sustain the inoculum throughout the testing period. The ingredients of the mineral medium are
detailed in Table 1. Salts with different amounts of hydration were able to be used and weights
were adjusted proportionately. High purity reagent grade water, free from inhibitory
concentrations of toxic substances (e.g., Cu2+ ions), was used for the preparation of the mineral
medium and all dosing stock solutions. Only one batch of water, which had been checked by
DOC analysis, was used. The water contained no more than 10% of the total carbon content
introduced by ethyl nicotinate. The pH of the test medium was 7.5 and was adjusted to 7.4 with
1 M hydrochloric acid.

2.6 Test Design
Each test unit (Figure 1) consisted of a 4-L glass bottle with a rubber stopper into which
one stainless steel needle with a Luer-Lok connection and two pieces of glass tubing were
inserted. Prior to test initiation, the test vessels were washed with detergent and rinsed with
water. The test vessels were then acid washed with 50% nitric acid and rinsed repeatedly with
reagent grade water. The stainless-steel needle was extended through the stopper into the testsolution serving as a sampling port for solution samples. A rubber policeman cap was used tocover the top of the sample port. The glass tubing provided the inlet and outlet ports for air
exchange. CO2-free air was pumped under positive pressure through a hydration flask before
entering the test system. The outlet port of each system was connected to two CO2
effluent gastraps, the first consisting of 200 mL of 0.2 N potassium hydroxide (KOH) and the second trap
containing 100 mL of 0.2 N KOH. The test vessels were identified with the study number,
replicate (A or B), and treatment type. Each test vessel was placed on a magnetic stir plate
located in a dark environmental chamber set to maintain a temperature of 22 ± 2 ºC.

2.7 Test Initiation
Seven test vessels were established: two for ethyl nicotinate, two inoculum blanks, one sodium
benzoate procedural control, and one toxicity control (Table 2). An additional vessel was
established in the same manner as the test suspension vessels, but was used only for pH
measurements and sampling for TIC and TC on day 0. This vessel was not attached to KOH
traps and was disposed of after pH, TC, and TIC determination.
On day -1, seven 4.0-L vessels (Figure 1) were established by adding either 2988 mL (for the
procedural and toxicity control) or 2991 mL mineral medium Table 1. A 6.0-mL aliquot of the
activated sludge inoculum and a 3.0-mL aliquot of the soil/sediment filtrate were added to each
vessel for a total volume of 2997 mL or 3000 mL per vessel. The seven test vessels were
attached to a CO2-free compressed air gas tank and aerated under positive pressure. The vessels
were mixed and purged with CO2-free air until day 0 to remove any residual inorganic carbon in
the test system prior to test initiation.

At test initiation (day 0), ethyl nicotinate replicate vessels (A and B) and additional sampling
vessel were dosed as described in Section 2.3. The total fortification was 10 mg C/L in the test
suspension vessels.

The inoculum blank control vessels only received inoculum and mineral medium. The toxicity
control vessel, which was fortified in the same manner as the test suspension vessels, also
received 3.0 mL of the 10 mg C/mL sodium benzoate stock solution for a total fortification of
20 mg C/L (test and reference substances). The sodium benzoate procedural control was
fortified with 3.0 mL of the sodium benzoate stock solution for a final concentration of
10 mg C/L. The pH test vessel was prepared in a similar manner to the test suspension vessels.
A summary of the dosing procedure is presented in Table 2.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

4.1 Test Conditions
The temperature in the environmental chamber was recorded daily, with the exception of
weekends, throughout the study. The temperature ranged from 20 to 21 ºC. The pH
measurements are presented in Table 3. The pH of the test medium was measured to be 7.50
upon preparation. The pH of the pH check vessel on day 0 was measured to be 7.78. The pH
values of the study vessels ranged from 7.73 to 7.93 at the end of the study on day 28.

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

ca. 83.69

St. dev.:

4.99

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

ca. 15.07

St. dev.:

4.94

Sampling time:

3 d

% Degradation 3

Parameter:

% degradation (CO2 evolution)

Value:

ca. 78.34

St. dev.:

3.42

Sampling time:

10 d

Details on results:

The total inorganic carbon measured in the KOH traps Table 4 was used to calculate the
cumulative CO2 evolved from the test vessels Table 5. The mean cumulative CO2
values from the ethyl nicotinate, procedural control, toxicity control, and blank control test vessels at day 28
were 48.57, 42.51, 73.05, and 17.88 mg/L, respectively. The cumulative net percent CO2
production (blank control values subtracted), or percent ultimate biodegradation, values for ethyl
nicotinate, procedural control, and toxicity control were calculated to be 83.69, 67.19, and
75.23% respectively (Table 6 and Figure 2). The cumulative net percent CO2
evolved from the procedural control was 62.28% of theoretical by day 13, thus meeting the “pass” criteria of the
test (reaching 60% or greater CO2 evolution within 14 days). This rapid biodegradation of
sodium benzoate confirmed the presence of an active microbial population and system integrity.

The toxicity control produced a cumulative net CO2 percentage of 66.80% by day 13,
demonstrating that ethyl nicotinate was not toxic to the inoculum as defined by
OECD Guideline 301B (i.e., < 25% on day 14 is considered inhibitory).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Based on the extent of CO2 evolution during this study, ethyl nicotinate can be classified as
readily biodegradable by the criteria set forth in OECD Guideline 301B since it did achieve
60% CO2 evolution within a 10-day window of reaching 10% biodegradation.