Ethyl 3,5-dichloro-4-hexadecyloxycarbonyloxybenzoate

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:

19 Nov 2019 - 20 Jan 2020

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

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"

Version / remarks:

2018

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: mixture of activated sludge and inoculum from soil

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge was freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage. The soil was obtained from standard soil Speyer 2.4 and Speyer 6S.
- Method of cultivation: Freshly obtained sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: In order to prepare soil as inoculum approximately 100 g of a 1:1 mixture of Speyer 2.4 soil and Speyer 6S soil was mixed with approximately 1 L of mineral medium. This mixture was put on a shaker for 30 minutes. After allowing the mixture to settle for 1 minute, the resulting suspension was decanted for use as inoculum.
- Pretreatment: The sludge was coarsely sieved (1 mm).
- Concentration of sludge: 9 mg/L suspended solids from sewage and 150 mg/L suspended solids from soil

Duration of test (contact time):

60 d

Initial conc.:

19.4 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301 guideline
- Additional substrate: High purity silica gel (Sigma-Aldrich, Steinheim, Germany) was added to each vessel at a concentration of 1 g/L (see Table 1 in 'any other information on materials and methods incl. tables').
- Test temperature: 22 - 23 °C
- pH: 7.3 - 7.7
- pH adjusted: no
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: 5 L brown coloured glass bottles connected in series to three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) to the exit aeration line of each test bottle.
- Number of culture flasks/concentration: 7
- Method used to create aerobic conditions: A mixture of oxygen (ca. 20 %) and nitrogen (ca. 80 %) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: Yes
- Details of trap for CO2 and volatile organics if used: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from air.
- Other: Before the start of the test (Day -1) mineral components, Milli-RO (reversed osmosis) water (ca. 80 % of final volume), and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

SAMPLING
- Sampling frequency: Titrations were made on Day 2, 5, 8, 12, 15, 19, 23, 28, 34, 40, 44, 49, 54, 61 for inoculum blank and test item. Titrations for procedural and toxicity control were made over a period of 14 days (last titration on Day 15).
- Sampling method: CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany). For each titration one to three CO2-absorbers were removed for titration. If applicable, remaining CO2-absorbers were moved in the direction of the test bottle. A corresponding number of new CO2-absorbers was placed at the far end of the series. On Day 60 the pH of respective test suspensions was measured and 2.5 mL of concentrated HCl (37 %, Merck) was added. Bottles were aerated overnight to drive off CO2. Final titration was performed on Day 61. For the procedural control and toxicity control, the pH was measured on Day 14 and 1 mL of concentrated HCl was added, followed by overnight aeration and a final titration on Day 15.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Positive Control: 1 vessel
- Toxicity control: 1 vessel

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

67

Sampling time:

60 d

Remarks on result:

other: result is the mean measured value of 7 replicates and based on ThCO2

Details on results:

Biodegradation of the test item in replicate test vessels ranged from 58 - 72% at day 60 based on ThCO2.

Results with reference substance:

Procedural control item was biodegraded by 83% within 14 days.

Table 1: Mean measured biodegradation (%)

	Biodegradation (%)
Day	Positive control^a	Toxicity control^b	Test item^c	Δ Min - Max^d
2	11	6	0	0
5	42	22	2	3
8	58	26	3	4
12	67	36	8	10
15	83*	50*	16	16
19			25	22
23			35	26
28			44	27
34			51	26
40			56	24
44			58	23
49			61	21
54			63	18
61			67^+	14

a = Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 215.3 mg CO2/5L.

b = Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and procedural control item: 436.8 mg CO2/5L (ThCO2 test item: 221.6 mg CO2/5L + ThCO2 sodium acetate: 215.3 mg CO2/5L).

c = Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item ranging from 218.9 – 222.1 mg CO2/5L within the replicates.

d = Absolute difference in biodegradation between test item bottles highest and lowest value.

* = CO2 measured on Day 15 is actually part of CO2 production of Day 14, since microbial activity was ended on Day 14 by addition of HCl.

+ = CO2 measured on Day 61 is actually part of CO2 production of Day 60, since microbial activity was ended on Day 60 by addition of HCl.

 

 

Table 2: Validity criteria for OECD 301 B

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	14%	Yes
Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).	83%	Yes
The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.	50%	Yes
The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.	< 5%	Yes
The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.	25.2 mg/L	Yes

 

Validity criteria fulfilled:

yes

Remarks:

See Table 2 in 'any other information on results incl. tables'

Interpretation of results:

other: ultimately biodegradable (i.e. >= 60% within 60 days)

Conclusions:

Calculations based on measurements performed during the test period revealed between 58% and 72% biodegradation of the test item for vessel A through G (7 replicates), based on ThCO2. Since average biodegradation of the test item in vessel A through G exceeded 60% within a test period of 60 days, the criterion for ultimate biodegradability was met. In the toxicity control, more than 25% biodegradation occurred within 14 days (50%, based on ThCO2). Therefore, the test item was considered not to inhibit microbial activity.

Description of key information

Ethyl 3,5-dichloro-4-hexadecyloxycarbonyloxybenzoate is ultimately biodegradable (>60% biodegradation within 60 days) (OECD 301B)

Additional information

The biological degradation of ethyl 3,5-dichloro-4-hexadecyloxycarbonyloxybenzoate (AF-366) in aerobic aqueous medium was evaluated in a study conducted according to OECD Guideline 301B and under GLP conditions. A mixture of activated sludge and soil was exposed to the test substance (19.4 mg/L, corresponding to 12 mg TOC/L) for 60 days at 22 - 23 °C. Sodium acetate was used as reference control and a toxicity control was included in the experimental set-up as well. The toxicity control and the positive control were incubated for 14 days. Calculations based on measurements performed during the test period revealed a mean degradation of 67% after 60 d (seven replicates), based on ThCO2. Since the average biodegradation of the test item was > 60% (67%) within a test period of 60 days, the criterion for ultimate biodegradability was met. This criterion was already met after an incubation of 49 d. In the toxicity control, the test item was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

Two additional studies are available which investigated the biodegradation of the target substance in aquatic environments. One study was conducted according to OECD Guideline 301B, under GLP conditions. Activated sludge was exposed to the test substance (10 and 20 mg/L) for 42 days at 20 °C. Sodium acetate was used as reference control and a toxicity control was included in the experimental set-up as well. After 28 days, 12% and 21% biodegradation was observed in the 10 mg/L and 20 mg/L treatment groups respectively, which increased to 21% and 34% at the end of the testing period (42 days) for the 10 mg/L and 20 mg/L groups, respectively. The results of the toxicity control showed that AF-366 did not inhibit the activity of the microorganisms. The other study was conducted according to Dutch Guidelines for the Determination of Biological and Chemical Oxygen Demand (NEN-3235.4 and NEN-3235-5.3), under GLP conditions. The BOD5 and BOD20 values had biodegradation values of 1.5 and 15%, respectively.

In conclusion, ethyl 3,5-dichloro-4-hexadecyloxycarbonyloxybenzoate was assessed as ultimately biodegradable (i.e. ≥60 % within 60 days).