Carbonotrithioic acid, bis(phenylmethyl) ester

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP compliant but difficult to conclude on biodegradability as there is toxicity to bacteria at the tested concentration (100 mg/L)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Flasks run: 2 test material, 1 blank, 1 inoculum control (sodium benzoate), 2 emulsifier control (Symperonic P103), 1 inhibition control (test material + sodium benzoate)

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Points of degradation plot (test substance):
0 % degradation after 10 d
0 % degradation after 28 d

Results with reference substance:

Points of degradation plot (reference substance):
40 % degradation after 1 d
53 % degradation after 5 d
60 % degradation after 10 d
73 % degradation after 15 d
72 % degradation after 20 d
78 % degradation after 28 d

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

According to the test conditions Dibenzyl trithiocarbonate shows no biodegradation, but inhibition of the inoculum is observed in the dedicated flask (no degradation of sodium benzoate when 100 mg/L of test substance is present).

Executive summary:

DIBENZYLTRITHIOCARBONATE is not readily biodegradable in the test conditions (manometric respirometry, concentration of test material: 100g/L)

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Chemical name: BLOCBUILDER DB EA50
Composition: Dibenzyl trithiocarbonate (active ingredient, CAS n° 26504-29-0) in ethyl acetate
Batch number: G07LFP0309
Date of reception in the test facility: 26 February 2018
Expiration date: 07 July 2018
Internal identification: 0004-18
Appearance: orange liquid
Water solubility: Dibenzyl trithiocarbonate: 0,34 mg/L (according to the SDS)
Relative density: Dibenzyl trithiocarbonate: 1.21 g/mL at 20°C (according to the EU registration dossier)
Storage conditions: well-ventilated place, ambient temperature and obscurity

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

INOCULUM
The inoculum was obtained on 26 June 2018 from the activated sludge of the biological wastewater treatment plant from Mourenx (France, 64) which handles predominantly domestic sewage.
On arrival at the laboratory, the sludge (around 1 L) was centrifuged at room temperature for 10 min at 1000 g; the solid sewage inoculant portion was re-suspended in dilution water in order to keep the concentration unchanged with respect to the sample. The inoculum was preconditioned 48 hours (aeration) until the flasks were inoculated.
Sub-samples of the homogenised sludge were dried in an oven at approximately 105°C and the suspended solids content determined to be 2.76 g/L.
The inoculum was not adapted to the test item before the biodegradation test.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Degradation is monitored by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas volume in the respirometer flask. Evolved carbon dioxide is absorbed in a solution of a suitable absorbent. The amount of oxygen taken up by the microbial population during biodegradation of the test substance (corrected for uptake by blank inoculum and other control if need be, run in parallel) is expressed as a percentage of ThOD.

STUDY DESIGN
The test was conducted using 16 bioreactors (500 mL nominal capacity, filled up to 250 mL):

¿ 2 for blank inoculum control (Fb1 & Fb2)
¿ 2 for reference control (Fc1 & Fc2)
¿ 2 for test item, directly added (Ft1 & Ft2, at 100 mg/L)
¿ 2 for test item, dissolved with the help of an emulsifying agent (Ft3 & Ft4, at 100 mg/L)
¿ 2 for solvent ethyl acetate control, directly added (Fs1 & Fs2, at 50 mg/L)
¿ 2 for solvent ethyl acetate control, dissolved with the help of an emulsifying agent (Fs3 & Fs4, at 50 mg/L)
¿ 2 for emulsifying agent control (Fa1 & Fa2)
¿ 1 for the toxicity control (Fi1).
¿ 1 for the toxicity control/emulsifying agent control (Fi2).

The toxicity of the solvent ethyl acetate was not assessed as it is already known to be no toxic on microorganisms at the tested concentration rate.
pH was measured in all bioreactors at the start and the end of the test period.

TEST SOLUTIONS PREPARATION

TEST ITEM (FT)
As the active ingredient has a low water solubility and dissolved at 50% in a volatile solvent (ethyl acetate), the test item falls into the category of a "difficult substance". Its dispersion in the bioreactor was assessed through 2 methods:
1. The test item was directly added to test bioreactors taking care to avoid volatilization (corresponding to Ft1 & Ft2)
2. Its dispersibility was improved by the use of an emulsifying agent as described in ISO 10634 standard (corresponding to Ft3 & Ft4)

This test protocol was designed in agreement with the Sponsor's Representative.
- Direct addition method: 25 µL of test item was pipetted and introduced in the test bioreactors filled up to 250 mL with the mineral medium to get the target addition rate for this study of 100 mg/L of test item (the test item density was not considered here).
- Emulsifying agent method: a test item stock solution was prepared using the emulsifying agent solution as prepared in the paragraph 3.3.4 below: 250 µL of test item was introduced in a 100 mL volumetric flask. The volume was made with the emulsifying agent solution. Both test item bioreactors contained 10 mL of this solution, corresponding to a final test item concentration of 100 mg/L.

ETHYL ACETATE (FS)
The ethyl acetate biodegradability was also measured to distinguish active ingredient/solvent biodegradability, and assessed according to both direct addition method and emulsifying agent method corresponding to the bioreactors Fs1/Fs2 and Fs3/Fs4 respectively.

- Direct addition method: 12.5 µL of ethyl acetate was pipetted and introduced in the test bioreactors filled up to 250 mL with the mineral medium to get the target addition rate for this study of 50 mg/L of ethyl acetate (the density of ethyl acetate was not considered here). This target rate is close to the ethyl acetate content in the test item (i.e. close to 50%)
- Emulsifying agent method: 12.5 µL of ethyl acetate was mixed with 10 mL of the emulsifying agent solution then introduced in the bioreactors filled up to 250 mL with the mineral medium.

REFERENCE ITEM SOLUTION (FC)
A parent solution of sodium benzoate in the dilution water at a concentration of 2.5 g/L was prepared (250.1 mg of reference item was weighed and poured in a 100 mL volumetric flask). The volume was made with the mineral medium. Both reference item bioreactors contained 10 mL of this solution, corresponding to a final reference item concentration of 100 mg/L.

EMULSIFYING AGENT SOLUTION (FA)
A 1.25 g/L stock solution was prepared from 312.5 mg of emulsifying agent Symperonic P103 in a 250 mL volumetric flask (the volume was made with the mineral medium). Both emulsifying agent bioreactors contained 10 mL of this solution.

ABIOTIC STERILE SOLUTION
No estimation of abiotic degradation was performed.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

-

Test performance:

-

Key result

Parameter:

% degradation (O2 consumption)

Value:

21.9

Sampling time:

28 d

Details on results:

The method of test item addition in bioreactors has no incidence on its biodegradability.
The maximum degradation level of the test item (Ft) was 26.5% at day 28. With regard to the oxygen uptake, this degradation level correspond to the ethyl acetate (Fs) biodegradability.

Notes: the test item expiry date was passed at the date of the definitive experiment. A new CoA with an extended expiry date was requested to the sponsor: the new analysis of batch G07LFP0309 has shown that the active ingredient dibenzyl trithiocarbonate content (44.31%) was lower than the content first determined and outside the sponsor specifications 47% - 53% as shown in the test item CoA supplied upon its reception. Since both analysis describe the actual test item composition, this deviation has no impact on the results or on the validity of the study. This deviations were considered to have not affected the outcome or the achievement of the study objectives.

Cumulated O₂uptake (mg/L)

Day	Fb1	Fb2	Fc1	Fc2	Ft1	Ft2	Ft3	Ft4	Fs1	Fs2	Fs3	Fs4	Fa1	Fa2	Fi1	Fi2
0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1	2	1.2	53.8	47.6	23	26.3	29.1	28	21.6	25.4	24.8	31.4	0	0	51.8	51.5
2	3.4	3.1	87.2	83.5	40.8	44.1	43.6	42.8	31	33.9	30.9	42.7	0	0	95.7	89.5
3	4.1	3.8	121	116.2	46	49.8	50.5	49.2	35	37.4	34.6	47.5	0	0	171.6	169.7
4	5.4	5.6	129.3	126.6	50	54.7	54.6	53.1	38.8	40.8	38.5	51.5	0	1.7	185.2	178.9
5	6.5	7.2	137.6	134.3	52.4	57.1	57.4	55.6	42.3	44.3	42.2	55.9	0	3.9	197.1	184.9
6	7.7	8.1	142.6	138.9	54.2	58.9	59.2	57.6	44.4	46.4	45	58.7	2.5	6.1	202.9	188.4
7	8.2	9.3	147.6	143.8	55.8	61.1	61.6	59.2	46.9	48.3	48.2	61.9	4.1	8	208.4	192.9
8	9.1	10.4	151.6	147.3	57	62.3	63.7	61.2	48.6	50.6	50.3	64.2	5.4	9.6	212.1	197.7
9	10.5	12.1	154.7	150.6	58.6	64.1	65.5	63.4	50.4	52	52.5	66.3	6.9	11.2	214.6	202.6
10	10.5	12.6	157.6	152.9	59	64.9	66.4	63.8	51.4	53.3	54	68	7.8	11.2	216.4	204.7
11	11.1	13.7	160.2	155.1	61.2	66.7	67.8	65.5	52.3	54.3	55.2	69.8	8.6	12.4	217.6	206.2
12	12.9	15.3	163	157.4	61.5	67.6	69.1	67.2	53.9	55.8	57.3	72	10.2	14	219.4	208.4
13	13.7	16.6	165.9	159.5	62.4	69.5	70.4	68.4	55.5	57.5	59.4	75	12.4	16.3	221.2	210.8
14	15	18.2	168.5	161.9	64.6	70.6	71.9	70.1	57.6	60.3	61.9	77.7	13.8	18	222.9	212.2
15	15.7	19.4	170.6	163.4	64.6	71.6	71.9	70.1	58	60.3	63.2	79.9	14.9	19.4	223.7	213.9
16	15.7	20	172.5	164.7	64.6	71.6	72.5	70.5	58.7	60.3	64.3	81.7	15.8	20.3	225	215.9
17	17.1	21.7	173.8	164.7	65.8	72.7	73.1	71.5	59.1	62	64.3	82.9	15.8	20.3	225	216.8
18	18.2	22.9	176.4	167.5	66.5	74	74	72.2	60.8	63.3	66.5	85.6	18	22.5	226.3	219.8
19	19.4	24.9	178.6	168.9	67.7	75.7	75.1	73.4	62.6	65.8	68	87.6	19.5	24.4	228	223
20	20.5	25.8	180.4	170.2	68.8	77	75.9	74.4	63.6	65.8	68.8	88.8	20.6	25	228.8	231.5
21	21.2	26.8	182	171.7	68.8	77	76.2	74.4	64.4	67.3	69.8	89.4	21.6	26.5	229.7	246.7
22	22.5	28	183.9	173.3	69.3	78	76.9	75	65.6	69.1	70.6	90.7	22.4	26.9	230.7	255.5
23	23.2	28.9	185.7	175.5	70.9	79.2	77.6	75.8	67	70.6	72.1	92.3	24	28.7	232.2	261
24	24.1	30.1	187.3	176.9	71.4	80.2	78.4	76.2	68.1	71.8	73.1	93.1	25	29.8	233.4	265.8
25	24.8	30.8	188.4	177.7	72.8	81.2	79	76.7	68.9	73.5	73.6	93.6	25.4	30.3	234.2	269.8
26	25.5	30.8	189.1	178.5	72.8	82.1	79.2	77.2	69.3	73.5	73.8	93.6	25.4	30.3	234.8	272.6
27	25.9	31.6	190.1	179.7	72.8	83.4	79.9	77.7	69.9	73.5	74.7	94.5	26.5	30.6	235.9	275.5
28	26.6	33	191	181.2	75.3	84.9	80.7	78.8	71	75.5	75.6	95	27.1	31.1	237	278.1

Biodegradation (%, reference control, Fi1 & Fi2 samples)

Day	Fc1	Fc2	Average Fc	Fi1	Fi2
0	0.0	0.0	0.0	0.0	0.0
1	31.3	27.6	29.5	13.4	13.8
2	50.4	48.2	49.3	24.7	23.9
3	70.3	67.4	68.8	44.8	45.3
4	74.3	72.7	73.5	48.0	47.6
5	78.5	76.5	77.5	50.8	48.9
6	80.9	78.6	79.7	52.1	49.2
7	83.3	81.1	82.2	53.3	49.9
8	85.1	82.6	83.9	54.0	50.8
9	86.1	83.6	84.8	54.3	51.7
10	87.7	84.8	86.3	54.7	52.1
11	88.7	85.7	87.2	54.8	52.3
12	89.4	86.0	87.7	54.8	52.4
13	90.5	86.6	88.6	55.0	52.5
14	91.2	87.2	89.2	55.1	52.4
15	91.9	87.5	89.7	55.1	52.6
16	92.8	88.1	90.5	55.3	52.8
17	92.7	87.2	89.9	54.9	53.1
18	93.5	88.2	90.9	55.0	53.3
19	93.9	88.1	91.0	55.0	53.7
20	94.4	88.3	91.3	54.9	55.7
21	94.8	88.7	91.7	54.9	59.5
22	95.2	88.9	92.0	54.9	61.7
23	95.8	89.7	92.8	55.1	62.7
24	96.2	89.9	93.0	55.1	63.7
25	96.4	90.0	93.2	55.1	64.6
26	96.6	90.2	93.4	55.2	65.4
27	96.8	90.6	93.7	55.3	66.0
28	96.8	90.9	93.8	55.3	66.5

Fc1: Reference control replicate 1

Fc2: Reference control replicate 2

Fi1: Toxicity control without emulsifying agent

Fi2: Toxicity control with emulsifying agent

Biodegradation (%, test item samples)

Day	Ft1	Ft2	Ft3	Ft4
0	0.0	0.0	0.0	0.0
1	10.3	11.9	14.0	13.5
2	18.1	19.7	21.0	20.6
3	20.2	22.1	24.3	23.7
4	21.4	23.7	25.9	25.1
5	21.9	24.2	26.7	25.8
6	22.3	24.5	26.4	25.6
7	22.6	25.2	26.7	25.6
8	22.7	25.3	27.0	25.8
9	22.8	25.4	27.2	26.2
10	22.8	25.7	27.4	26.1
11	23.5	26.1	27.6	26.5
12	22.8	25.7	27.4	26.5
13	22.7	26.2	27.0	26.0
14	23.1	26.0	26.9	26.1
15	22.6	26.0	26.3	25.5
16	22.5	25.9	26.2	25.2
17	22.3	25.6	26.5	25.7
18	22.1	25.7	25.9	25.0
19	21.9	25.8	25.6	24.8
20	22.0	25.9	25.6	24.8
21	21.6	25.5	25.1	24.2
22	21.2	25.4	25.1	24.2
23	21.6	25.6	24.7	23.8
24	21.3	25.6	24.5	23.5
25	21.7	25.7	24.6	23.5
26	21.5	26.0	24.7	23.7
27	21.2	26.3	24.7	23.7
28	21.9	26.5	24.8	23.9

Ft1 & Ft2: test item, direct addition method

Ft3 & Ft4: test item, emulsifying agent method

pH recorded

Treatment	Flask	Day 0	Day 29
Blank control	Fb1 & Fb2	7.4	7.4
Reference control	Fc1 & Fc2	7.4	7.7
Test item	Ft1 & Ft2	7.4	7.4
	Ft3 & Ft4	7.4	7.4
Ethyl acetate	Fs1 & Fs2	7.4	7.4
	Fs3 & Fs4	7.4	7.3
Emulsifying agent control	Fa1 & Fa2	7.4	7.3
Toxicity control	Fi1	7.4	7.9
	Fi2	7.4	7.7

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

According to the OECD 301 guideline, the pass levels for ready biodegradability is 60% of ThOD for respirometric methods. This pass value has to be reached in a 10-d window within the 28-d period of the test; the 10-d window begins when the degree of biodegradation has reached 10% ThOD and must end before day 28 of the test.

In this study the pass level 60% of ThOD was not reached (biodegradation max is 26.5%). As a consequence the test item BLOCBUILDER DB EA50 was not readily biodegradable under the conditions of the test.

Executive summary:

The study was conducted in accordance with procedures outlined in OECD Guideline 301F (1992). The ready biodegradability of BLOCBUILDER DB EA50 was assessed over a 28 days period (data were recorded over a 29 days period, due to laboratory organisation).

 

As the active ingredient has a low water solubility and is dissolved at 50% in a volatile solvent (ethyl acetate), the test item falls into the category of a "difficult substance". Its dispersion in the bioreactor was assessed through 2 methods:

 

1.    The test item was directly added to test bioreactors taking care to avoid volatilization (corresponding toFt1 & 2)

2.    Its dispersibility was improved by the use of an emulsifying agent as described in ISO 10634 standard (corresponding toFt3 & 4)

 

The ethyl acetate biodegradability was also measured to distinguish active ingredient/solvent biodegradability, and assessed according to both direct addition and emulsifying agent methods. The toxicity of the test item and the emulsifying agent was assessed.

The test was conducted at a nominal loading rate of 100 mg/L for the test item and 50 mg/L for ethyl acetate (as contained at 50% in the test item). A total of 16 bioreactors were used. This test protocol was designed in agreement with the Sponsor's Representative.

 

The results of the test were as follows:

% biodegradation at 28 day
Test item	Reference item (mean of replicates)	Toxicity Control (test item)	Toxicity Control (test item & emulsifying agent)
21.9% to 26.5%	93.8%	55.3%	66%

The maximum degradation level of the test item was 26.5% at day28 (with regard to the oxygen uptake, this degradation level correspond to the ethyl acetate biodegradability).The test item was thus not readily biodegradable under the conditions of the test as it failed to achieve a transition from 10% to 60% degradation in a 10-day window during the 28 days test.

The definitive test met the validity criteria of the test guideline detailed as follows:

§ the difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20% (min.: 21.9%, max.: 26.5%)

§ the percentage degradation of the reference compound has reached the pass levels by day 14: 89.2% (mean of replicates)

§ the biodegradation observed in both toxicity control bioreactors was not less than 25% occurred within 14 days: 55.1% and 52.5%

§ the oxygen uptake of the inoculum blank was not greater than 60 mg/L in 28 days: 26.6 mg/L and 33.0 mg/L

§  the pH value was not outside the range 6-8.5: min 7.3, max 7.9

Description of key information

DBTTC: no conclusion on biodegradation can be reached (inhibition of the inoculum observed)

DBEA50: not readily biodegradable (% biodegradation measured after 28d: from 21.9 to 26.5%).

Additional information

In the first reliable study following the OECD Guideline 301F performed on dibenzyl trithiocarbonate (DBTTC) by Gancet (2005), no conclusion on biodegradation can be reached as inhibition of the inoculum has been observed.

 

In the second reliable study following the OECD Guideline 301F performed on dibenzyl trithiocarbonate in solution in ethyl acetate (DBEA50 ; about 50% v/v) by Chastenet (2018), the test item can be considered as not readily biodegradable (% biodegradation measured after 28d: from 21.9 to 26.5%).