Carbonotrithioic acid, bis(phenylmethyl) ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

The minimal recorded test temperatures during the pre-test was 20.8°C that is outside the range of 21-24°C ± 2°C specified in the study plan. This deviation was considered to have not affected the outcome or the achievement of the study objectives.

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: 1.21 g/mL at 20°C
Storage conditions: well-ventilated place, ambient temperature and obscurity

Analytical monitoring:

yes

Details on sampling:

Extra replicates (two replicates vessels at each test concentration except for the control) were added to the definitive test in order to perform additional samplings for analysis at 24 hour intervals during the exposure period (one replicate was sacrificed at T24h, the second one at T48h)
Samples of 200µL were taken from all test concentrations, the control and the satellite vessels at the start of the test and after 24 hours, 48 hours and 72 hours of exposure. Inoculated samples were previously filtered then analysed according to the analytical procedure
Time between sampling and measurement is approximately 15 – 30 min.

Vehicle:

no

Details on test solutions:

TEST SOLUTION
Based on the results of the pre-test, 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.
The range of concentrations was obtained from dilutions of the saturated stock solution prepared as follows: a known volume of test item (40 µL) was poured into a volumetric flask, the volume was then made up to 4 L with test medium.
The solution was manually shacked, place in an ultrasonic bath during 15 minutes then kept under high speed stirring at ambient temperature during approximately 65 hours with a magnetic stir bar. Due to the presence of undissolved particles, this solution was centrifuged (3000 rpm, 10 minutes) before dilution. No auxiliary substance was used.
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 appropriate procedures were thus chosen in accordance with the Sponsor's Representative to design the test conditions: glass bottles of 120 mL nominal capacity filled up to 115 mL, tightly closed with bungs and without headspace were used to avoid volatilization.

TEST MEDIUM
Osmotically filtered water was used to prepare the dilution water.
As the use of a sealed exposure system in the algal growth inhibition test will result in culture growth being limited by CO2 depletion and increasing pH, the test medium was completed with a buffer solution ("Hepes medium" at 1 mM with NaHCO3 at 0.6 g/L) and sodium bicarbonate.
Chemical reagents used for the preparation of dilution water were of “analytical grade”.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK).

Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline.

The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use.

Three days before the start of the exposure, two pre-cultures were prepared by inoculating sufficient cells from the algal stock culture to the growth medium to obtain a low cell density, e.g. 5.10E3 cells/mL to 10E4 cells/mL for pre-culturing, in order to maintain exponential growth until the start of the test. The pre-cultures were incubated under the same conditions as those used for the test cultures. Only one of the two pre-cultures was used to inoculate the test flasks for the study. The second one was only used if the first one was damaged.

At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 5.10E3 or 10E4 cells/mL as recommended in the OECD 201 guideline.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

-

Test temperature:

Pre-test: range from 20.8 to 21.6°C (mean: 21.2°C)
Definitive test: range from 21.9 to 22.5°C (mean: 22.2°C)

pH:

Pre-test: range from 8.3 to 9.7
Definitive test: range from 8.1 to 9.5

Dissolved oxygen:

Pre-test: range from 7.2 mg/L to OFL*
Definitive test: range from 8.4 mg/L to OFL*

*OFL : Saturation of the measurement device

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Pre-test concentrations expressed as:
- % saturated stock solution (% v/v): 10, 25, 50 and 100
- geometric mean of measured concentrations (µg/L): 0.49 ; 1.42 ; 2.57 ; 4.30


Definitive test concentrations expressed as:
- % saturated stock solution (% v/v): 3.9 ; 5.9 ; 8.8 ; 13.2 ; 19.8 ; 29.6 ; 44.4 ; 66.7 ; 100.0
- geometric mean of measured concentrations (µg/L): 0.25 ; 0.30 ; 0.54 ; 0.76 ; 0.99 ; 1.44 ; 1.96 ; 5.87 ; 8.64

Details on test conditions:

The incubation was conducted in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 22 ± 1°C; flasks were continuously shaken with a rotation speed at around 100 rpm and constantly illuminated by fluorescent tubes.
Test vessels were made of glass bottles (120 mL capacity) capped with cellulose bungs. They were filled with a volume of 50 mL.
Dissolved oxygen and pH measurements were carried out in non-inoculated solution at T0 and inoculated solution at T72. An increase in the pH is observed, this may be associated to consumption of the dissolved CO2 due to the growth of algae. In addition, it is known that tightly closed test flasks lead to an increase of O2 due to the absence of gas exchanges. Despite this, it is not expected to have affected the outcome of the study as all validity criteria were still met.
Furthermore, the light intensity was measured as follows:
- Pre-test: range from 134 to 138 µE·m-2 s-1
- Definitive test: range from 111 to 113 µE·m-2 s-1

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 8.64 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: Maximum concentration achievable after 72h

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.35 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Details on results:

PRE-TEST

The appearance of the test solutions was visually checked: at the beginning of the test the solutions were found to be slightly cloud. Algae were exposed to this raw samples but an additional analysis was performed on samples after centrifugation (3000 rpm, 10 minutes) to confirm the presence of undissolved test item particles. The results obtained shows non-homogeneous samples with high levels of test item concentrations, meaning that test solutions need to be centrifuged. No precipitation was observed at the end of the test.
Chemical analysis of test samples taken at 0 and 72h indicated that concentrations of the test item were not maintained over the 72h test period (i.e. deviation > 20%). The instability of the test item was thus confirmed over the test period.
Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm. Only slight distortion was observed.
The control culture had an exponential growth at the expected rate throughout the test.
Algal growth was strongly inhibited at all tested concentrations: mean of cells number at 72h range from 1.9 to 7.104 cells/mL. This excessive toxicity is probably due to the high level of undissolved test item particles in the test medium. In addition, it was demonstrated that acetonitrile has significant effects on the algae growth.


DEFINITIVE TEST

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation was observed at the end of the test.
Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm. Only slight distortion was observed.
Chemical analysis of test samples indicated that concentrations of the test item were not maintained over the 72h test period (i.e. deviation > 20%). Chemical analysis of the test item showed the instability of the substance over the 72-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.
The control culture had an exponential growth at the expected rate throughout the test.

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 every two months by performing an algal growth inhibition test on potassium dichromate. The latest value of ErC50 obtained in June 2018 was 1.35 mg/L.
For information, according to ISO 8692 standard, 72h-ErC50 obtained from an inter-laboratory exercise on potassium dichromate was in the range 0.65 to 1.73 mg/L.

Reported statistics and error estimates:

STATISTICAL ANALYSIS / DATA TREATMENT

EC10 and EC50 values as well as the 95% confidence intervals were then determined using the software ToxRat 3.2.1 according to the recommendations of the guideline.
As there was evidence that the concentration of the substance being tested were not satisfactorily maintained within ± 20% of the measured initial concentrationthroughout the test, analysis of the results was based on geometric mean concentration during exposure or on models describing the decline of the concentration of the test substance.
Note: calculations were performed with Microsoft Excel. The values presented in the report have been rounded. Thus, hand calculations may differ slightly from those presented due to rounding.

Definitive test results

Growth Function	72h-EC10(µg/L)	95% confidence level		72h-EC50 (µg/L)	95% confidence level
		Lower limit	Upper limit		Lower limit	Upper limit
Growth rate	2.35	1.78	3.10	>8.64	ND	ND
Yield	0.71	0.49	1.04	3.74	2.37	5.83

Algal cell density in the definitive test (.10⁴cells/mL)

Treatm. [µg/L]		Control	0.25	0.3	0.54	0.76	0.99	1.44	1.96	5.87	8.64
0 h	a	1	1	1	1	1	1	1	1	1	1
	b	1	1	1	1	1	1	1	1	1	1
	c	1	1	1	1	1	1	1	1	1	1
	d	1
	e	1
	f	1
Mean:		1	1	1	1	1	1	1	1	1	1
Std.Dev.:		0	0	0	0	0	0	0	0	0	0
n:		6	3	3	3	3	3	3	3	3	3
CV:		0	0	0	0	0	0	0	0	0	0
24 h	a	4.5	6.5	4.1	3.9	5	3.6	2.6	4.1	2	2.8
	b	3.9	6	3.5	3.7	5.2	3.7	2.8	3.8	2	2.2
	c	4.2	5.8	4	4.9	4.3	3.1	2.7	3.4	2.1	3.7
	d	3.1
	e	3.2
	f	3.7
Mean:		3.8	6.1	3.9	4.2	4.9	3.5	2.7	3.8	2	2.9
Std.Dev.:		0.55	0.32	0.34	0.63	0.46	0.31	0.11	0.36	0.08	0.75
n:		6	3	3	3	3	3	3	3	3	3
CV:		14.6	5.3	8.7	15.1	9.4	8.9	4.2	9.5	3.9	25.8
48 h	a	26.6	35.7	30.3	34.4	30.9	31.8	22.9	23	7.9	11
	b	27.2	31	29.9	32.5	29.9	32.7	24.8	26.6	9.1	9.3
	c	31	31.7	31.1	35.3	28.4	28.5	24.1	20.7	9.2	12.6
	d	29.4
	e	35.4
	f	30.2
Mean:		30	32.8	30.4	34.1	29.8	31	23.9	23.4	8.7	11
Std.Dev.:		3.16	2.52	0.64	1.43	1.25	2.2	0.92	2.98	0.73	1.69
n:		6	3	3	3	3	3	3	3	3	3
CV:		10.5	7.7	2.1	4.2	4.2	7.1	3.9	12.7	8.4	15.4
72 h	a	115.2	125.9	124	117.6	117.2	121.7	83.2	89.1	36	43
	b	120.6	118	114.5	121.3	112.2	114.1	93.1	98.3	40.3	34.3
	c	123.5	128.1	111.5	124.7	105.3	111.7	94.9	83.8	38.9	45.8
	d	139.6
	e	134.7
	f	130.4
Mean:		127.3	124	116.7	121.2	111.6	115.8	90.4	90.4	38.4	41
Std.Dev.:		9.21	5.33	6.51	3.54	5.95	5.22	6.32	7.35	2.2	5.97
n:		6	3	3	3	3	3	3	3	3	3
CV:		7.2	4.3	5.6	2.9	5.3	4.5	7	8.1	5.7	14.5

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	1.71	1.32	1.52	1.52	0.20	13
5.9	2.31	1.83	2.10	2.08	0.24	11
8.8	3.45	3.05	3.07	3.19	0.23	7.1
13.2	5.13	4.46	4.43	4.67	0.40	8.5
19.8	8.31	6.78	6.39	7.16	1.01	14
29.6	11.4	9.86	9.83	10.3	0.87	8.4
44.4	15.8	14.5	14.1	14.8	0.88	5.9
66.7	24.7	24.6	25.4	24.9	0.46	1.8
100.0	35.5	33.2	33.8	34.2	1.2	3.4

Test item determination at T24h (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	ND	ND	ND	NA	NA	NA
3.9	0.51	0.55	0.55	0.54	0.02	4.2
5.9	*	0.77	0.66	0.72	0.08	11
8.8	1.25	1.25	1.23	1.24	0.01	0.94
13.2	1.87	2.01	2.12	2.00	0.13	6.4
19.8	2.37	2.74	2.68	2.60	0.20	7.7
29.6	3.99	4.27	4.38	4.22	0.20	4.8
44.4	6.14	6.24	6.45	6.28	0.16	2.5
66.7	9.72	9.48	9.87	9.69	0.20	2.0
100.0	17.1	16.5	16.5	16.7	0.38	2.3

*outlier value

Test item determination at T48h (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	ND	ND	ND	NA	NA	NA
3.9	< QL	< QL	< QL	< QL	NA	NA
5.9	0.20	< QL	< QL	< QL	NA	NA
8.8	0.32	0.44	0.37	0.38	0.06	16
13.2	0.64	0.65	0.64	0.64	0.01	0.8
19.8	0.89	0.99	0.87	0.92	0.06	6.8
29.6	1.89	1.83	1.67	1.80	0.11	6.2
44.4	2.77	2.82	2.98	2.86	0.11	3.9
66.7	5.28	5.23	5.20	5.24	0.04	0.8
100.0	8.31	8.49	9.58	8.79	0.69	7.8

Test item determination at 72h (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	< LD	< LD	< LD	< LD	NA	NA
5.9	< LD	< LD	< LD	< LD	NA	NA
8.8	< LD	< LD	< LD	< LD	NA	NA
13.2	< LD	< LD	< LD	< LD	NA	NA
19.8	< LD	< LD	< LD	< LD	NA	NA
29.6	< LD	< LD	< LD	< LD	NA	NA
44.4	< LD	< LD	< LD	< LD	NA	NA
66.7	0.81	0.99	1.01	0.94	0.11	12
100.0	1.01	1.15	1.16	1.11	0.08	7.4

< DL (0.055 µg/L): concentration lower than the Detection Limit

< QL (0.184 µg/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 measured concentrations(µg/L)								Exposure conc. (µg/L)**
	T0	T24	Deviation T0/T24	T48	Deviation T24/T48	T72	Deviation T48/T72	Deviation T0/T72
Control	<DL	<DL	NA	<DL	NA	<DL	NA	NA	NA
3.9	1.52	0.54	-64.5	0.092*	-83.0	0.055*	-40.2	-96.4	0.25
5.9	2.08	0.72	-65.4	0.092*	-87.2	0.055*	-40.2	-97.4	0.30
8.8	3.19	1.24	-61.1	0.38	-69.4	0.055*	-85.5	-98.3	0.54
13.2	4.67	2	-57.2	0.64	-68.0	0.055*	-91.4	-98.8	0.76
19.8	7.16	2.6	-63.7	0.92	-64.6	0.055*	-94.0	-99.2	0.99
29.6	10.3	4.22	-59.0	1.8	-57.3	0.055*	-96.9	-99.5	1.44
44.4	14.8	6.28	-57.6	2.86	-54.5	0.055*	-98.1	-99.6	1.96
66.7	24.9	9.69	-61.1	5.24	-45.9	0.94	-82.1	-96.2	5.87
100.0	34.2	16.7	-51.2	8.79	-47.4	1.11	-87.4	-96.8	8.64

< DL (0.055 µg/L): concentration lower than the Detection Limit

< QL (0.184 µg/L): concentration lower than the Quantification Limit

NA: Not Applicable

*When test item measured concentration is < DL, DL value was used

 When test item measured concentration is < QL, half QL value was used

**Concentrations extrapolatedas geometric mean of measured concentrations

Validity criteria fulfilled:

yes

Conclusions:

The results are reported as growth rate and yield inhibitions.
The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined.


Growth Function 72h-EC10 (µg/L) 95% confidence level 72h-EC50(µg/L) 95% confidence level
Lower limit Upper limit Lower limit Upper limit
Growth rate 2.35 1.78 3.10 >8.64 ND ND
Yield 0.71 0.49 1.04 3.74 2.37 5.83

ND: Not Determined due to mathematical reasons or inappropriate data

Executive summary:

This study was designed to determine the effects of the test item on the growth of Pseudokirchneriella subcapitatain a 72-hour test according to the OECD 201 Guideline.

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 appropriate procedures were thus chosen in accordance with the Sponsor's Representative to design the test conditions: glass bottles of 120 mL nominal capacity filled up to 115 mL, tightly closed with bungs and without headspace were used to avoid volatilization.

In the absence of reliable data regarding the test item behavior in an aqueous medium a preliminary test was performed in order to assess the solubility and stability of the test item in the test medium with the objective to design the definitive test protocol in accordance with the Sponsor's Representative: the definitive test conditions were designed from the results obtained during the pre-test and also form the conclusions deducted from a Daphnia acute toxicity test performed at the test facility.

 

The definitive test solutions were prepared from dilutions of a saturated stock solution. Algae cells were exposed to the following series of dilutions: 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. 72h) were not satisfactorily maintained within ± 20% of the measured initial concentration that's why geometric means of measured concentrations were used as exposure concentrations.The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined:

Growth Function	72h-EC10(µg/L)	95% confidence level		72h-EC50 (µg/L)	95% confidence level
		Lower limit	Upper limit		Lower limit	Upper limit
Growth rate	2.35	1.78	3.10	>8.64	ND	ND
Yield	0.71	0.49	1.04	3.74	2.37	5.83

ND: Not Determined due to mathematical reasons or inappropriate data

 

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

 - The biomass in the control cultures increased exponentially by a factor of 127.3 within the 72-hour test period (required: at last 16-fold)

 - The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was equal to 25.5% (required: < 35%)

 -  The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 1.5% (required: < 7%)

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on test solutions:

For the range finding test a stock solution of the test item was prepared 3 days before the beginning of the test (time 0), by mixing 50 mg (41.6 µL) 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 HLVP 047000 at 0.45 µm. The parent solution obtained was clear and colourless. Dilutions were made in order to obtain a series of 5 concentrations (0.1, 1, 5, 10, 50 mg/L) of DIBENZYL TRITHIOCARBONATE in dilution water. In order to improve toxicity figures, test was voluntarily carried out at concentrations above water solubility (saturated solutions).

For the definitive test a stock solution of the test item was prepared 20 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 HLVP 047000 at 0.45 µm. The solution obtained was clear and colourless.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

Dilution water is prepared according to the protocol described in Annex 3 of the OECD guideline 201.

Test temperature:

23 ± 1°C

pH:

Concentrations (nominal)mg/L pH
To T72h
0 (BI) 7.93 7.98
0 (T) - 9.24
5 8.0 8.75

Dissolved oxygen:

Concentrations (nominal)mg/L Dissolved 02 (mgIL)
To T72h
0 (BI) 9.0 8.7
0 (T) - 10.4
5 8.3 9.7

Salinity:

Dilution water is prepared according to the protocol described in Annex 3 of the OECD guideline 201.

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 (4.3 µgIL).

Details on test conditions:

The incubation was performed in a phytoculture cabinet that aliows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C; flasks were continuously shaken with a rotation at 20 rpm and constantly illuminated by 8 fluorescent tubes between 6,000 and 10,000 lux (Mazdafluor, white industry 33).
The study was performed using 120 mL glass boules stoppered with cellulose bungs. Filling volume: 50 mL

Osmotically filtered water was used to prepare the dilution water (composition according to GL 201). Chemical reagents used for the preparation of dilution water were of "analytical grade".

Physico-chemical parameters were measured using a METTLER TOLEDO 345 pH meter for measurement of pH (LPX028) and with a WTW OXI 538 oxymeter (LPX027) for dissolved oxygen measurement.

Determination of algae concentration was carried out using cytofluorimetry with a Cytofluor 2350 device in the preliminary test. In the definitive test, cells are numbered either with a with a tell counter (Beckman Coulter Z2).

In the preliminary test, algae were exposed under static conditions to a series of 5 concentrations (0.1, 1, 5, 10, 50 mg/L) of DIBENZYL TRITHIOCARBONATE in dilution water (saturated solutions). The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.
Test solutions were inoculated with aigae as described in § 3.1. AII concentrations were prepared as triplicates. Flasks were stoppered with cellulose bungs and placed in a phytoculture cabinet (Strader DCS Pulsar).

An inoculated control flask (labelled T) was prepared and incubated under the same conditions, with no test item. This was used for determining algae growth. A non-inoculated biank (labelled BI) containing only dilution water and test item was also prepared and incubated.

After 24, 48, and 72 h of incubation, a volume of 200 µL was sampled from each test flask, pipetted into a quartz microplate. Fluorescence was then determined using a cytofluorimeter and by comparison with a calibration range, celi density was determined, according to the measured fluorescence.
Dissolved 02 and pH were measured in the control and the highest concentration, in non-inoculated flask at the beginning of the test and in an inoculated flask at the end of the test.

Definitive test (Iimit test):
An inhibition was observed at the higher nominal concentration (50 mg/L). This is due to the tact that this concentration is much higher than the solubility limit and that the toxicity observed is probably due to the hydrophilic toxic impurities occurring in the test item. Therefore, in order to improve toxicity figures but keeping in mind solubility issues and minimizing artefact related to impurities, test was voluntarily carried out at one concentration above water solubility (saturated solutions).
It was thus decided to run a limit test at 5 mg/L (nominal concentration) which is already far above the solubility in water (i.e. 0.34 mg/L).

Number of replicates: Test flasks and blanks were prepared in triplicate. Six controls were prepared.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Details on results:

Definitive test - Average percentage inhibition of cell growth (lAi) and growth rate (lµi)
Concentration IAi Iµi
(nominal)
mg/L (%) (%)
0 0.00 0.00
5 -20.95 -5.91

Results with reference substance (positive control):

The OECD 201 method does flot require to test a reference item to be tested simultaneously to the assay on the test item. However the sensitivity of the test system and the methodology are evaluated every two months by performing an afgal growth inhibition test on sodium dichromate. The nearest values of ErC50 and EbC50 obtained on June 12, 2008 were respectively 1.60 mgIL and 0.57 mglL.

Conclusions:

With a nominal loading rate of 5 mg/L no toxicity was observed (either cell growth or growth rate).

Executive summary:

The determination of the growth rate inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to dibenzyl trithiocarbamate for a duration of 72h was assessed according to the OECD Guideline 201.

Algae were exposed to a 5 mg/L of dibenzyl trithiocarbamate in dilution water.

The concentrations of test item causing a 50% reduction in growth rate were estimated: ErC50 > 5 mg/L.

Description of key information

DBTTC:

- 72h-ErC50: no effect up to the water solubility

- 72h-ErC10: no effect up to the water solubility

 

DBEA50

- 72h-ErC50 > 8.64 µg/L (maximum concentration achievable after 72h)

- 72h-ErC10 = 2.35 µg/L

Additional information

In the first reliable study following the OECD Guideline 201 performed on dibenzyl trithiocarbonate (DBTTC) by Gancet (2009) with nominal concentrations above the water of solubility:

- the concentration leading to a 50% reduction in growth rate is 72h-ErC50 > 0.34 mg/L (no effect above the water solubility)

- the concentration leading to a 10% reduction in growth rate is 72h-ErC10 > 0.34 mg/L (no effect above the water solubility)

 

In the second reliable study following the OECD Guideline 201 performed on dibenzyl trithiocarbonate in solution in ethyl acetate (DBEA50 ; about 50% v/v) by Chastenet (2018)

- the concentration leading to a 50% reduction in growth rate is 72h-ErC50 > 8.64 µg/L (measured concentration ; maximum concentration achievable after 72h)

- the concentration leading to a 10% reduction in growth rate is 72h-ErC10 = 2.35 µg/L (measured concentration)