Tetramethylthiuram monosulphide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None.

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the control on 4 occasions throughout the test, on fresh and old solutions for each occasion. Samples were analysed on the same day as sampling.

Vehicle:

no

Details on test solutions:

See the details on test conditions section.

Test organisms (species):

Daphnia magna

Details on test organisms:

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

Test animals were fed a diet of 0.1 - 0.2 mg of carbon per daphnid per day, in the form of a suspension of the algal strains Chlorella vulgaris (25%) and Pseudokirchneriella subcapitata (75%), except during the initial three days of culture when a slightly lower ration was given. The concentrated algal cell suspension was prepared by removing and centrifuging aliquots of algal culture and resuspending the algal pellets in small volumes of dilution medium.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

165-170 mg/L as CaCO3 (min-max)

Test temperature:

20.3 - 20.6°C (min-max)

pH:

7.4 - 9.1 (min-max)

Dissolved oxygen:

8.6 - 14.5 mg/L (min-max)

Salinity:

Freshwater.

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal concentrations: 0 ; 0.0032 ; 0.01 ; 0.032 ; 0.1 ; 0.32 ; 1 mg/L
Arithmetico-geometric mean concentrations: 0 ; 0.0021 ; 0.0061 ; 0.0207 ; 0.0706 ; 0.1914 ; 0.5476 mg/L

Details on test conditions:

STUDY DESIGN
The primary objective of this study was to determine the toxicity of TMTM to daphnia reproduction. A secondary objective was to investigate if the photolytic degradation product(s) of TMTM were toxic to daphnia reproduction and compare their toxicity to that of TMTM. To do so, additional flasks pre-exposed to light for a sufficient time to obtain 100% degradation products (“aged flasks”) were inoculated (introduction of parent daphnia) and incubated at the same time and under the same conditions as the "fresh flask". Pre-exposure time to light was 24 hours. New aged flask were prepared every day to be used 24 hours later. As the volatility of the potential degradation products was unknown, the experiment was carried out in a way that prevented chemical evaporation (glass bottles tightly closed with bungs and without headspace in both fresh and aged flasks). All vessels used in the experiment were strictly identical, as flask characteristics may affect composition of penetrating light and thus photodegradation rate.

TEST ITEM SOLUTIONS PREPARATION
In order to prepare "aged flasks", a first test item stock solution (at 1 mg/L nominal concentration) was prepared: a known volume mass of test item (around 40 mg) was poured into a volumetric flask, the volume was then made filled up to 4000 mL with the test medium. This solution was kept under high speed stirring at ambient temperature during a few minutes with a magnetic stir bar , then it was incubated in the climatic chamber within test conditions (temperature and light intensity) during 24 hours (i.e. until start of the test for the first batch or next medium renewal). No filtration was necessary

DEFINITIVE REPRODUCTION TEST
Daphnids were exposed to a serie of test item concentrations prepared from successive dilutions of each fresh and old stock solutions:

- Test item nominal concentrations for "Aged flasks": 0.1 and 1 mg/L
- Test item nominal concentrations for "Fresh flasks": 0.0032, 0.01, 0.032, 0.10, 0.32 and 1.00 mg/L

Ten replicate vessels were prepared at each concentration including the control group, except at the nominal concentration 0.010 mg/L where a male neonate was introduced at Day 0 and was thus discarded from results and statistical analysis.

At the start of the experiment one D. magna neonate, less than one day old, was added to each vessel. Daphnia were fed as described above. The total test period was 21 days. All test solutions (including control) were renewed every day (including weekends). Samples for analysis were taken from all test concentrations and the control on 4 occasions throughout the test, on fresh and old solutions:

- at days 1 (fresh and aged solutions) and 2 (old solutions)
- at days 9 (fresh and aged solutions) and 10 (old solutions)
- at days 15 (fresh and aged solutions) and 16 (old solutions)
- at days 20 (fresh and aged solutions) and 21 (old solutions)

ENVIRONMENTAL CONDITIONS
Test vessels containing D. magna were incubated at a temperature ranging from 18°C to 22°C but remaining constant within ± 2°C during the test. A light cycle of 16h light and 8h dark was in operation throughout the test, provided by LED light (light intensity around 800 lux, light spectrum 400-700 nm, Lumen 3450 lm). Test vessels were made of clear glass tubes (120 mL capacity) tightly closed with bungs and without headspace. The control treatment was maintained under the same conditions as the treated groups, except it did not contain any test item. No auxiliary substance was used.

OBSERVATIONS
Every day, all parent animals were observed for immobilisation by gentle agitation of the test vessel. Any immobile animal within 15 seconds following agitation was considered as dead and discarded. The surviving parental Daphnia were transferred into fresh control or test media in a second set of vessels by means of a wide bore glass pipette. From the day of the first brood, all neonates (dead and alive), unhatched eggs or carapaces present in the expired media were counted daily and discarded. Any other abnormal observation was also recorded and reported. pH and dissolved oxygen were measured in the fresh and old solutions in one of the vessels of each concentration group with surviving daphnids. The total hardness used to prepare test solutions was determined using a Hardness test kit. The temperature was continuously recorded throughout the test, in an abiotic flask maintained under the same experimental conditions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

FRESH FLASKS
Chemical analysis of the test item showed the instability of the substance over a 24-hours test period. Based on these results, the exposure concentrations were based on the arithmetic mean of the four geometric means of the concentrations measured in new and old media over 24-h periods. Immobility of parent daphnia was higher than 90% in the four highest treatments. There were no immobile Daphnia magna in the control group and in the two lowest treatments (NOEC = 0.0061 mg/L for parent immobility). In addition, there were no difference of living neonate production per surviving parent between the two lowest treatments and the control group (NOEC = 0.0061 mg/L). Because only two treatments survived and no effect was observed in these treatments, no ECx could be determined because no regression model was applicable.

AGED FLASKS
Chemical analysis of test samples showed that the test item was not completely degraded and thus the toxicity observed could not be completely attributed to the test item degradation products. All adult Daphnia magna were found immobile a few days after start the test in all aged flasks. As a consequence, the effects of the test item degradation products on the reproductive capacity of Daphnia magna could not be assessed.

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 nearest value of 24h-EC50 obtained on March 2016 was 0.64 mg/L. For information, ISO 6341 has a validity criteria specifying that EC50 must be in the range 0.6 to 2.1 mg/L.

Reported statistics and error estimates:

The arithmetic mean of the four geometric means of the concentrations measured in new fresh/aged and corresponding old media over 24-h periods were used for the estimation of ECx and NOEC/LOEC values. Statistical analysis was conducted on the following variables: number of living neonates produced by surviving parent, parent mobility and age at first brood. All statistical analysis were conducted using the Software ToxRat 2.10.

Nominal conc. (mg/L)		Test item concentration (mg/L)			Geometric mean (mg/L)	Arithmetic mean (mg/L)
		Sampling time	Fresh	Old
Fresh flask	0.0032	D1	0.0034	0.0018	0.00247386	0.0021
		D9	0.0036	0.0013	0.00216333
		D15	0.0031	0.0012	0.00192873
		D20	0.0032	0.0012	0.00195959
	0.01	D1	0.0099	0.005	0.00703562	0.0061
		D9	0.0104	0.0036	0.00611882
		D15	0.0098	0.0035	0.00585662
		D20	0.0097	0.003	0.00539444
	0.032	D1	0.0363	0.0165	0.02447346	0.0207
		D9	0.0367	0.0115	0.02054386
		D15	0.032	0.013	0.02039608
		D20	0.0313	0.0096	0.01733436
	0.1	D1	0.1099	0.0454	0.07063611	0.0706
	0.32	D1	0.3472	0.1055	0.19138861	0.1914
	1	D1	1.0553	0.2842	0.54764611	0.5476
Aged flask	0.1	D1	0.0271	0.0089	0.01553029	0.0155
	1	D1	0.2764	0.0846	0.15291645	0.1529

		Fresh flask (mg/L)							Aged flask (mg/L)
		Control	0.0021	0.0061	0.021	0.071	0.19	0.55	0.0155	0.1529
a	liv. off.	63	135	149	0	0	0	0	0	0
	dead off.	0	2	2	0	0	0	0	0	0
	egg	0	1	5	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead
b	liv. off.	143	123	120	20	0	0	0	0	0
	dead off.	1	2	1	1	0	0	0	0	0
	egg	0	2	0	0	0	0	0	0	0
	dead	Alive	Alive	Alive	alive	Dead	Dead	Dead	Dead	Dead
c	liv. off.	140	128	134	0	0	0	0	0	0
	dead off.	2	3	2	0	0	0	0	0	0
	egg	0	1	1	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead
d	liv. off.	152	187	0	0	0	0	0	0	0
	dead off.	1	2	0	0	0	0	0	0	0
	egg	1	0	0	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead
e	liv. off.	137	179	162	0	0	0	0	0	0
	dead off.	0	4	4	0	0	0	0	0	0
	egg	0	0	3	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead
f	liv. off.	154	139	157	0	0	0	0	0	0
	dead off.	0	5	3	0	0	0	0	0	0
	egg	0	0	0	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead
g	liv. off.	160	162	153	0	0	0	0	0	0
	dead off.	1	5	1	0	0	0	0	0	0
	egg	0	1	1	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead
h	liv. off.	186	208	184	0	0	0	0	0	0
	dead off.	3	3	0	0	0	0	0	0	0
	egg	0	0	1	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead
i	liv. off.	176	182	164	0	0	0	0	0	0
	dead off.	0	2	3	0	0	0	0	0	0
	egg	0	1	0	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead
j	liv. off.	177	134	167	0	0	0	0	0	0
	dead off.	0	0	3	0	0	0	0	0	0
	egg	0	0	2	0	0	0	0	0	0
	dead	Alive	Alive	Alive	Dead	Dead	Dead	Dead	Dead	Dead

	Fresh flask (mg/L)							Aged flask (mg/L)
	Control	0.0021	0.0061	0.021	0.071	0.19	0.55	0.0155	0.1529
Total of living offspring	1488	1577	1390	20	0	0	0	0	0
Total of dead offspring	8	29.7	19	1	0	0	0	0	0
Total of egg	1	6	13	0	0	0	0	0	0
Total of dead parent	0	0	0	9	10	10	10	10	10
Mean of living offspring produced per parent	148.8	157.7	1154.4	20	n.a	n.a	n.a	n.a	n.a
Standard Deviation	34.5	29.7	19.0	ND	n.a	n.a	n.a	n.a	n.a

Validity criteria fulfilled:

yes

Remarks:

At the end of the test: 0% mortality of the parent animals ; mean number of living neonates produced per parent animal surviving is =148.8 ; CV of the mean number of living neonates produced per parent animal survivingt is 23%

Conclusions:

- Mean number of neonates produced per survivor - Fresh flasks (test item assessment)
21d-EC10: nd
21d-EC50: nd
NOEC: 0.006 mg/L
LOEC: 0.021 mg/L

- Mean number of neonates produced per survivor - Aged flasks (degradation products assessment)
21d-EC10: nd
21d-EC50: nd
NOEC: nd
LOEC: nd

- Parent mobility - Fresh flasks (test item assessment)
21d-EC10: nd
21d-EC50: nd
NOEC: 0.006 mg/L
LOEC: 0.021 mg/L

- Parent mobility - aged flasks (degradation products assessment)
21d-EC10: nd
21d-EC50: nd
NOEC <0.015 mg/L
LOEC =0.015 mg/L

- Age at first brood - Fresh flasks (test item assessment)
21d-EC10: nd
21d-EC50: nd
NOEC: 0.006 mg/L
LOEC: 0.021 mg/L

- Age at first brood - aged flasks (degradation assessment)
21d-EC10: nd
21d-EC50: nd
NOEC: nd
LOEC: nd

nd: not determined for mathematical reasons or inappropriate data

Executive summary:

This study was designed to determine the effects of Tetramethylthiuram monosulphide on Daphnia magna reproduction and survival in a 21 days test according to the OECD 211 Guideline (2012). A secondary objective was to investigate if the photolytic degradation product(s) of TMTM were toxic to daphnia reproduction and compare their toxicity to that of TMTM. To do so, additional flasks preexposed to light for a sufficient time to obtain 100% degradation products (“aged flasks”) were inoculated (introduction of parent daphnia) and incubated at the same time and under the same conditions as the "fresh flask". Pre-exposure time to light was 24 hours. New aged flask were prepared

every day to be used 24 hours later. As the volatility of the potential degradation products was unknown, the experiment was carried out in a way that prevented chemical evaporation (glass bottles tightly closed with bungs and without headspace in both fresh and aged flasks). All vessels used in the experiment were strictly identical, as flask characteristics may affect composition of penetrating light and thus photodegradation rate. The total test period was 21 days. Nominal test item concentrations of fresh solutions were 0.0032, 0.01, 0.032, 0.10, 0.32 and 1.00 mg/L. Nominal test item concentration of aged solutions were 0.1 and 1 mg/L. The test solutions (including control) were renewed daily throughout the test (including weekends). Ten replicate test vessels were prepared for the control and the test concentrations. A single juvenile Daphnia magna (<24 hours old selected by filtration) was added to each test vessel (except at the nominal concentration 0.010 mg/L where a male neonate was introduced at Day 0 and was thus discarded from statistical analysis). On each renewal occasion, parental animals were transferred into new test medium (“fresh” or “aged”). Any juveniles remaining in old test media were

counted and the number of juveniles produced per parental animal on any day during the 21-day study duration was recorded. Chemical analysis of test samples showed the instability of the test substance between two medium renewals. Chemical analysis from aged samples also showed the test item was not completely degraded by pre-exposure to light (27% test item remained). Thus the toxicity observed could not be entirely attributed to the test item degradation products. Since (i) all parent daphnia died at 0.1 and 1 mg/L nominal concentrations in the aged flasks and (ii) all parent daphnia died at a lower nominal concentration in the fresh flasks (0.032 mg/L), it would not have been possible to rank the toxicities of the test item and its degradation products anyway. In particular, it would not have been possible to assess if the degradation products were at least as toxic as the test item. Therefore, as a worst case, the exposure concentrations over 24 hours in the “fresh” solutions were estimated on the basis of geometrical means of measured concentrations. The mean exposure concentration over the

whole experiment was then calculated as the arithmetical mean of the four geometrical means. NOEC, LOEC, EC10 and EC50 values on day 21 were calculated to be as follows:

- Mean number of neonates produced per survivor - Fresh flasks (test item assessment)

21d-EC10: nd

21d-EC50: nd

NOEC: 0.006 mg/L

LOEC: 0.021 mg/L

- Mean number of neonates produced per survivor - Aged flasks (degradation products assessment)

21d-EC10: nd

21d-EC50: nd

NOEC: nd

LOEC: nd

nd: not determined for mathematical reasons or inappropriate data

Parent mobility - Fresh flasks (test item assessment)

21d-EC10: nd

21d-EC50: nd

NOEC: 0.006 mg/L

LOEC: 0.021 mg/L

Parent mobility - aged flasks (degradation products assessment)

21d-EC10: nd

21d-EC50: nd

NOEC <0.015 mg/L

LOEC =0.015 mg/L

- Age at first brood - Fresh flasks (test item assessment)

21d-EC10: nd

21d-EC50: nd

NOEC: 0.006 mg/L

LOEC: 0.021 mg/L

Age at first brood - aged flasks (degradation assessment)

21d-EC10: nd

21d-EC50: nd

NOEC: nd

LOEC: nd

nd: not determined for mathematical reasons or inappropriate data

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.006 mg/L

Additional information

The chronic toxicity of tetramethylthiuram monosulphide to Daphnia magna was assessed through several studies for which the followings results were determined:

• LC50-21d: 0.164 mg/L 


• NOEC-21d: 0.006 mg/L

The first data is mentioned in the HPV dossier Thiuram, but as the study belongs to another company, the tox data is considered as supporting data. Thus, the "key data" is NOEC-21d: 0.006 mg/L