Carbonotrithioic acid, bis(phenylmethyl) ester

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

TEST ITEM

Chemical name: BLOCBUILDER DB EA50
Composition: active ingredient: Dibenzyl trithiocarbonate (synonym: carbonotrithioic acid, bis(phenylmethyl) ester, 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

Analytical monitoring:

yes

Details on sampling:

Sampling at 0 and 48h

Vehicle:

no

Details on test solutions:

The definitive test solutions were prepared from dilutions of a saturated stock solution prepared at a 10 mg/L of loading rate.
A known volume of test item (20 µL) was poured into a volumetric flask, the volume was then made up to 2000 mL with test medium.
The solution was manually shacked, place in a ultrasonic bath during 15 minutes then kept under high speed stirring at ambient temperature during approximately 24 hours with a magnetic stir bar. Due to the presence of undissolved particles, this solution was centrifuged (3000 rpm, 10 minutes) before dilution.
The definitive test was conducted at the following series of dilutions of the saturated stock solution: 3.9, 5.9, 8.8, 13.2, 19.8, 29.6, 44.4, 66.7 and 100% v/v. Four vessels were prepared at each test concentration and the control group; each vessel contained 5 daphnids.
The pH of the test solutions were not adjusted.
Test solutions were not renewed throughout the 48h test period (static test).

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organisms used for this study were Daphnia magna Straus (Cladocera, Crustacea), clone 5, bred within the laboratory by acyclical parthenogenesis in a synthetic medium. Daphnia magna cultures were fed with a mixture of the microalgae Chlorella vulgaris and Pseudokirchneriella subcapitata. The neonates used were less than 24h old at test initiation (selected by filtration) and were not first brood progeny.
Test animals were not fed during the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

total hardness (in the control group at the test initiation): 250 mg CaCO3/L

Test temperature:

from 20.3 to 20.4°C

pH:

from 7.9 to 8.1

Dissolved oxygen:

from 8.0 to 8.6 mg O2/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal concentrations (% saturated stock solution): 0 ; 3.9 ; 5.9 ; 8.8 ; 13.2 ; 19.8 ; 29.6 ; 44.4 ; 66.7 ; 100.0
Measured concentrations (µg/L): 0 ; 2.0 ; 3.0 ; 4.0 ; 6.0 ; 8.6 ; 14.3 ; 19.4 ; 34.5 ; 58.5

Details on test conditions:

Culture and test vessels containing D. magna (20 daphnids per tested concentration for the definitive test) were incubated with a photo period of 8 hours of darkness per 24h at a temperature ranging from 18°C to 22°C but remaining constant within ± 1°C during the test.
As the active ingredient is dissolved at 50% in a volatile solvent (ethyl acetate), the test item falls into the category of a "difficult substance" and thus the test vessels (made of clear glass bottles of around 40 mL capacity) were completely filled (i.e. without headspace) and tightly closed to avoid volatilization.
The control treatment was maintained under the same conditions as the treated groups, except it did not contain any test item. As decided following the pre-experiments, no auxiliary substance was used.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

16.01 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Details on results:

Chemical analysis of test samples taken at 0 and 48h indicated that measured concentrations of the test item were not maintained over the 48h test period (i.e. deviation >20% between T0 and T48h). As a consequence, the stability of the substance was not confirmed over the test period, the exposure concentrations were thus based on the geometric mean of measured concentrations.

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 deviation was considered to have not affected the outcome or the achievement of the study objectives.

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated monthly by performing an acute Daphnia magna toxicity test on potassium dichromate. The most recent values of 24h-EC50, obtained on August 2018, was 24h-EC50 = 0.77 mg/L
The ISO 6341 norm has a validity criterion specifying that the reference item EC50 must be in the 0.6 to 2.1 mg/L range.

Reported statistics and error estimates:

Statistical analysis was performed with the software ToxRatPro Version 3.2.1. A Probit analysis using linear max. likelihood regression was statistically considered to be more adapted to the raw data obtained.

Test item determination at T0 (Definitive test)

Test solution (% saturated stock solution)	Test item concentration(µg/L)			Mean (µg/L)	SD (µg/L)	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	NA	NA	NA
3.9	3.07	3.58	3.65	3.43	0.32	9.2
5.9	4.52	5.59	5.29	5.13	0.55	11
8.8	6.68	*	*	6.68	NA	NA
13.16	9.96	11.0	11.5	10.8	0.77	7.1
19.73	15.7	16.9	17.7	16.8	1.0	6.1
29.60	22.3	25.7	26.1	24.7	2.0	8.4
44.40	35.3	38.2	36.6	36.7	1.5	4.0
66.72	56.3	58.0	57.3	57.2	0.9	1.6
100.00	82.0	90.0	99.7	90.6	8.8	9.8

*analytical device deficiency

< DL (0.183 mg/L): concentration lower than the Detection Limit

< QL (0.610 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

Test item determination at 48h (Definitive test)

Test solution (% saturated stock solution)	Test item concentration(µg/L)			Mean (µg/L)	SD (µg/L)	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	NA	NA	NA
3.9	1.23	1.23	1.17	1.21	0.03	2.7
5.9	1.62	1.88	1.66	1.72	0.14	8.2
8.8	2.28	2.33	2.48	2.36	0.10	4.4
13.2	3.12	3.23	3.55	3.30	0.22	6.7
19.8	4.20	4.13	4.83	4.39	0.38	8.7
29.6	7.39	9.09	8.42	8.30	0.85	10
44.4	9.97	10.1	10.9	10.3	0.5	5.0
66.7	21.3	19.8	21.4	20.8	0.9	4.2
100.0	31.7	44.5	37.2	37.8	6.4	17

< DL (0.183 mg/L): concentration lower than the Detection Limit

< QL (0.610 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

Test item determination – Concentrations used for statistical analysis (Definitive test)

Test solution (% saturated stock solution)	Test item concentration(µg/L)		Deviation of 48h measured conc. from 0h (%)	Exposure conc. (µg/L)*
	T0	T48h
Control	< LD	< LD	NA	NA
3.9	3.43	1.21	-64.7	2.0
5.9	5.13	1.72	-66.5	3.0
8.8	6.68	2.36	-64.7	4.0
13.2	10.8	3.30	-69.4	6.0
19.8	16.8	4.39	-73.9	8.6
29.6	24.7	8.30	-66.4	14.3
44.4	36.7	10.3	-71.9	19.4
66.7	57.2	20.8	-63.6	34.5
100.0	90.6	37.8	-58.3	58.5

< DL (0.183 mg/L): concentration lower than the Detection Limit

< QL (0.610 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

*Concentrations extrapolated as geometric mean of measured concentrations

Measured pH and dissolved oxygen (Definitive test)

Test solution (% saturated stock solution)	pH		Dissolved O2 (mg/L)
	0h	48h	0h	48h
Control	8.1	7.9	8.6	8.2
3.9	8.0	8.0	8.6	8.1
5.9	8.1	7.9	8.6	8.1
8.8	8.0	7.9	8.6	8.0
13.2	8.1	7.9	8.6	8.0
19.8	8.0	7.9	8.6	8.1
29.6	8.0	7.9	8.5	8.1
44.4	8.0	8.0	8.5	8.1
66.7	8.0	8.0	8.5	8.1
100.0	8.0	8.0	8.5	8.2

Cumulative immobilisation (Definitive test)

Test solution (% saturated stock solution)	Total daphnids at T0	Immobile daphnids		Mobility inhibition at 48h(%)
		24h	48h
Control	20	0	0	0.0%
3.9	20	0	0	0.0%
5.9	20	0	0	0.0%
8.8	20	0	0	0.0%
13.2	20	0	0	0.0%
19.8	20	0	0	0.0%
29.6	20	2	4	20%
44.4	20	11	20	100.0
66.7	20	14	19	95%
100.0	20	11	20	100.0

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 measured for the test item is 16.01 µg/L

Executive summary:

This study was designed to determine the effects of the test item to Daphnia magna over a 48h period with static conditions, according to the OECD 202 Guideline.

 In the absence of reliable data regarding the test item behavior in an aqueous medium many pre-experiments were performed in order to determine the solubility and stability of the test item in the test medium. Various test combinations were designed to check the stirring method and duration to achieve maximum solubility in the test medium with the objective to design the definitive test protocol. 

 The definitive test solutions were prepared from dilutions of a saturated stock solution prepared at a 10 mg/L of loading rate.Daphnia were exposed to the following series of dilutions of this saturated stock solution: 3.9, 5.9, 8.8, 13.2, 19.8, 29.6, 44.4, 66.7 and 100% v/v.

The test item concentrations measured at the end of the test period (i.e. 48h) were not satisfactorily maintained within ± 20% of the measured initial concentration that's why geometric means of measured concentrations were used to determine 48h-EC10 and 48h-EC50 (all results are µg/L):

 

			95% confidence level
			Lower limit	Upper limit
Definitive test (static test)	48h-EC10	11.40 µg/L	ND	ND
	48h-EC50	16.01 µg/L	ND	ND

ND: Not Determined due to mathematical reasons or inappropriate data

 

 

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

 

- Immobilisation in the control did not exceed 10% at the end (0%)

- Dissolved oxygen concentrations remained above 3 mg/L over the test periods

 

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Storage of samples: samples were analysed without storage. Samples treatment: no treatment

Vehicle:

no

Details on test solutions:

A stock solution of DIBENZYL TRITHIOCARBONATE diluted in water was prepared at the beginning of the test (time 0) by mixing 5 mg of DIBENZYL TRITHIOCARBONATE in one litre of dilution water. The mixture was kept under stirring during 18 hours. The solution was then filtered through HAWP 04700 Millipore filter (0.45 pm) allowing to obtain a limpid solution. The solution obtained was clear and colourless.

In the preliminary test, daphnids were exposed under static conditions to a series of 5 concentrations (5, 2.5, 1, 0.5 and 0.1 mg/L) of DIBENZYL TRITHIOCARBONATE in dilution water (composition detailed in Annex 1). The toxic effect measured during the assay was the inhibition mobility over a time period of 48 hours.

Approximately 90% of the final volume of the test solution was added into test flasks. Five Daphnia magna aged from 6 to 24 hours were added into each test flask, which were then totally filled with the test solution (30 mL). Two replicates were prepared for each concentration. Two control flasks were prepared under the same conditions, with no test item. Test flasks were incubated in darkness at a temperature between 20.0 °C and 20.5 °C.

After 48 hours of incubation, mobile Daphnia magna were counted.
Dissolved 02 and pH were measured at the highest tested concentration and in the control. Results of the preliminary test served to define the concentration range used in the definitive test.

As results of the preliminary test showed no toxicity even at the higher nominal concentration, it was decided to run a limit test at 5 mg/L (nominal concentration, saturated solution) which is a concentration exceeding water solubility of the substance (ca 0.34 mg/L). This has been done in order to improve toxicity figures.

A stock solution of the test item was prepared 18 hours before the beginning of the test (time 0) by mixing 5 mg of DIBENZYL TRITHIOCARBONATE in 1 litre of dilution water.
At the end of this stirring period, which corresponded to the day on which the test was started, the solution was filtered on a Millipore membrane HAWP 047000 at 0.45 µm. The parent solution obtained was clear, colourless, slightly odorous.

The test was performed at the nominal concentration: 5 mg/L. Number of replicates: 4

Approximately 90% of the final volume of the test solution was added into test flasks. Five Daphnia magna aged from 6 to 24 hours were added into each test flask, which were then totally filled with the test solution (30 mL). 4 replicates were prepared for each concentration. 4 control flasks were prepared under the same conditions, with no test item. Test flasks were incubated in darkness at a temperature of 20.9 °C.

After 24 hours of incubation, mobile Daphnia magna were counted and flasks replaced under incubation. At time 48 hours mobile Daphnia magna were counted.

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

no

Hardness:

Chemical reagents used for the preparation of dilution water are of « analytical grade ». Dilution water is prepared according to the protocol described in standard NF EN 1SO 6341:
The following stock solutions are prepared:
CaCI2, 2H2O: 11.76 g per litre of water, Merck Ref. 1.02382.5000
MgSO4, 7H2O: 4.93 g per litre of water, Merck Ref. 1.05886.1000
NaHCO3: 2.59 g per litre of water, Prolabo Ref. 27778.293
KCI: 0.23 g of per litre of water, Prolabo Ref. 26764.232
Dilution water is obtained by adding 25 mL of each of the stock solutions in a final volume of 1 litre and then aerating the solution until oxygen saturation.
Sum of Ca and Mg ions in dilution water is of 2.5 mmol/L and ratios of Ca/Mg et Na/K are 4 and 10 respectively.

pH:

Nominal concentrationsmg/L
To T48h
0 7.97 7.75
5 7.90 7.68

Dissolved oxygen:

Nominal concentrationsmg/L Dissolved 02 (mgIL)
To T48h
0 9.1 8.4
5 8.2 7.5

Salinity:

according to the protocol described in standard NF EN 1SO 6341

Nominal and measured concentrations:

Loading rate the definitive (limit) test : 5 mg/L
Concentrations of DIBENZYL TRITHIOCARBONATE in the definitive test
NominalConcentration(mg/L) Measured in non inoculated solutions
Initial(mg/L) Final(mg/L)
0 (T) < DL < DL
5 < DL < DL
< DL : concentration lower than the Detection Limit of the analytical method (12.4 µgIL).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

> 0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: yes
- Mortality of control: no

Validity criteria fulfilled:

yes

Conclusions:

With a nominal loading rate of 5 mg/L no toxicity was observed.

Executive summary:

The acute toxicity (inhibition of mobility) of the test item DIBENZYL TRITHIOCARBAMATE for a duration of 48 hours was assessd accordinf to the OECD Guideline 202.

Daphnia were exposed to nominal concentrations of Dibenzyl trithiocarbamat exceeding its water solubility.

No toxic effect was observed after a 48h-exposure at a nominal loading rate of 5 mg/L.

Description of key information

DBTTC - 48h-EC50: no effect up to the limit of solubility

DBEA50 - 48h-EC50 = 16.01 µg/L

Additional information

In the first reliable study following the OECD Guideline 202 performed on dibenzyl trithiocarbonate (DBTTC) by Gancet (2009), the concentration leading to a50% immobilisation of the daphnids is 48h-EC50 > 0.34 mg/L (no effect up to the limit of solubility)

In the second reliable study following the OECD Guideline 202 performed on dibenzyl trithiocarbonate in solution in ethyl acetate (DBEA50 ; about 50% v/v) by Chastenet (2018), the concentration leading to a50% immobilisation of the daphnids is 48h-EC50 = 16.01 µg/L (measured concentration)