thiourea; thiocarbamide

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of the test was to develop and evaluate different bioassays to assess the hazard of chemicals to higher plants in relation to Level 2 of the German Chemicals Act:
In different assays reduction of germination and growth was tested:

A) 3d in-vitro test according to Moewus (1949) and Neururer (1975) with garden cress (Lepidium sativum) to assess the inhibition of germination and seedling development.
Nine concentrations (4 replicates) of thiourea covering 'no reaction' to 'total inhibition' of germination including untreated controls were tested using two sheets of moistened (5 ml distilled water conatining thiourea) filter paper covering the bottom of a Petri dish. Twenty-five untreated cress seeds were placed on the paper. Dishes were closed and stored in the dark at 25 °C for 72 h.

B) Growth test with oats (Avena sativa) and turnips (Brassica rapa) for 10 and 14 days, respectively, in soil and nonsorptive substrate (vermiculite).
Plants were cultivated in self-watering pots with glass fiber wicks (see Stadler and Pestemer, 1980) containing 70 g dry sandy loam soil (organic carbon 1.3 %, sand 54.3 %, silt 35.7 %, clay 9.9 %, max. water-holding capacity 23.2 g/ 100 g soil) or 70 ml vermiculite (mesh size 1-3 mm; same volume and water content as pots containing soil).
Thiourea (9 concentrations in 4 replicates) was dissolved in water and mixed into the soil and stored over night at 4 °C to maintain a sorption equilibrium. For pots with vermiculite the thiourea dissolved in water was sucked into the pots through the glass fiber wick of the self-watering system.
Seven plants per pot were transferred as 5 to 6 day old seedlings and placed in a greenhouse at 20-22 °C during day (additional lighting with HQL lamp; 16 h light/day), and 15 °C at night. Pots were watered with a combination of Penningsfold-summer and Hoagland-A-Z solution or with a commercial hydroponic fertilizer. After 10 and 14 days the shoot fresh weight was recorded for turnips and oats, respectively.

C) Long-term (8 week) bioassay with oats in hydroponic culture was developed to examine whether extended exposure has an effect (dose-response-time relationship).
Concentrations that showed slight or no effects in bioassays (A) and (B) were used to determine the influence of long-term exposure. Oats were cultivated in Quickpot plates (2 cm diam., 4 x 5 pots) that were placed in 1 L plastic containers containing a nutrient-chemical solution. Six plants per container were transplanted. The nutrient-chemical solution was renewed once a week to maintain constant chemical and nutrient concentrations. The total fresh weight (shoot and root) of all plants per container was recorded weekly, by weighing the whole system. Thiourea was applied at 8 concentrations in 6 replicates.

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

Not applicable.

Vehicle:

yes

Details on preparation and application of test substrate:

For all details for bioassays A, B, and C please refer to field "Principles of methods if other than guideline".

Species:

Lepidum sativum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Garden cress
- Plant family: Brassicaceae (Cruciferae)
- Variety: Sperling's Mega Grossblättrige
- Used in bioassay A

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Oats
- Plant family: Poaceae (Gramineae)
- Variety: Fabian
- Used in bioassay B and C

Species:

Brassica rapa

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Turnip
- Plant family: Brassicaceae (Cruciferae)
- Variety: rapa, Runde Weiße Rotköpfige
- Used in bioassay B

Test type:

other: germination and growth

Study type:

laboratory study

Substrate type:

other: Filter paper (bioassay A); artificial soil and non-sorptive substrate (bioassay B); quickpot plates in nutrient-chemical solution (bioassay C)

Limit test:

no

Total exposure duration:

8 wk

Remarks:

Bioassay A): total exposure duration 3 d; bioassay B): total exposure duration 10-14 d; bioassay C): total exposure duration 8 wk

Post exposure observation period:

Not applicable.

Test temperature:

Bioassay A: 25 °C
Bioassay B: 20-22 °C (day), and 15 °C (night)
Bioassay C: no data

pH:

Bioassay A: no data
Bioassay B: soil: 6.1, vermiculite; no data
Bioassay C: no data

Moisture:

Bioassay A: filter paper with 2 ml distilled water
Bioassay B: no data
Bioassay C: no data

Details on test conditions:

For all details for bioassays A, B, and C please refer to field "Principles of methods if other than guideline"

Nominal and measured concentrations:

Nominal concentratiosn:
- Bioassay A: 0-10,000 mg/L
- Bioassay B: 0-1000 mg/kg
- Bioassay C: 0-200 mg/L

Reference substance (positive control):

yes

Remarks:

2,6-dichlorobenzonitrile (germination inhibitor)

Species:

Lepidum sativum

Duration:

3 d

Dose descriptor:

other: EC5

Effect conc.:

ca. 100 other: mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Key result

Species:

Lepidum sativum

Duration:

3 d

Dose descriptor:

EC50

Effect conc.:

ca. 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Species:

Brassica rapa

Duration:

10 d

Dose descriptor:

other: EC5

Effect conc.:

21.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks on result:

other: Bioassay B, soil experiment

Key result

Species:

Brassica rapa

Duration:

10 d

Dose descriptor:

EC50

Effect conc.:

52.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks on result:

other: Bioassay B, soil experiment

Species:

Brassica rapa

Duration:

10 d

Dose descriptor:

EC50

Effect conc.:

67.3 other: mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks on result:

other: Bioassay B, vermiculite experiment

Species:

Avena sativa

Duration:

14 d

Dose descriptor:

other: EC5

Effect conc.:

ca. 30 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks on result:

other: Bioassay B, soil experiment

Key result

Species:

Avena sativa

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

339.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks on result:

other: Bioassay B, soil experiment

Species:

Avena sativa

Duration:

8 wk

Dose descriptor:

other: EC5

Effect conc.:

ca. 10 other: mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks on result:

other: Result of bioassay C.

Species:

Avena sativa

Duration:

8 wk

Dose descriptor:

other: EC5

Effect conc.:

ca. 3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks on result:

other: The reported EC5 of ca. 10 mg/L was converted into mg/kg soil dw using the same equations as for calculating the PNECsoil from PNECwater (EPM).

Details on results:

Bioassay B)
The bioavailability of thiourea was calculated to be almost 100 % (comparison of the concentration-response curves from soil and vermiculite experiments). Additional analysis (Pestemer, unpublished data) of the mineral nitrogen level (Nmin) in the substrates revealed a higher degree of Nmin in soil compared to vermiculite or also agar-agar (in both media very low microbial activity), indicating faster degradation of thiourea in soil, which could result in a metabolite with higher phytotoxicity.

Bioassay C):
The EC50 and EC5 decreased within the first four weeks of the experiment from 170 to 30 mg/L, and from 22 to 12 mg/L, respectively.

Results with reference substance (positive control):

Bioassay B): EC50 in soil (10-d, Brassica rapa) = 0.69 mg/kg
EC50 in vermiculite (10-d, Brassica rapa) = 0.18 mg/L

Reported statistics and error estimates:

The data (germination and growth) were analysed by non-linear regression (Günther et al., 1989). From the logistic curves obtained, the EC5 based on growth reduction was derived. For the long-term bioassay C the weekly logistic regression curves were derived and EC values were compared.

Validity criteria fulfilled:

not specified

Conclusions:

The most sensitive results in the different bioassays are as follows:
- Bioassay A): Lepidium sativum, 3-d EC50 = ca. 1000 mg/L
- Bioassay B): Brassica rapa, 10-d EC50 = 52.1 mg/kg soil dw
- Bioassay C): Avena sativa, 8-wk EC5= ca. 3 mg/kg soil dw

Executive summary:

The effect of thiourea on the seedling germination and growth of the monocot Avena sativa (oats) and the dicots Brassica rapa (turnip) and Lepidium sativum (garden cress) was studied in three different bioassays.

Bioassay A:

A 3-d in-vitro test according to Moewus (1949) and Neururer (1975) with Lepidium sativum was conducted to assess the inhibition of germination and seedling development. Nine concentrations (4 replicates) of thiourea covering 'no reaction' to 'total inhibition' of germination including untreated controls were tested using moistened filter paper.

Bioassay B:

Growth test, conducted with oats (Avena sativa) and turnips (Brassica rapa) for 10 and 14 days, respectively, in soil and non-sorptive substrate (vermiculite). Plants were cultivated in self-watering pots containing 70 g dry sandy loam soil or 70 ml vermiculite. Thiourea was tested at nine concentrations in 4 replicates. After 10 and 14 days the shoot fresh weight was recorded for turnips and oats, respectively.

Bioassay C:

A long-term (8 week) bioassay with oats in hydroponic culture was developed to examine whether extended exposure has an effect (dose-response-time relationship) on germination and growth. Concentrations that showed slight or no effects in bioassays (A) and (B) were used to determine the influence of long-term exposure. Oats were cultivated in Quickpot plates that were placed in 1 L plastic containers containing a nutrient-chemical solution. Six plants per container were transplanted. The nutrient-chemical solution was renewed once a week to maintain constant chemical and nutrient concentrations. The total fresh weight (shoot and root) of all plants per container was recorded weekly, by weighing the whole system. Thiourea was applied at eight concentrations in six replicates.

The most sensitive results in the different bioassays are as follows:

- Bioassay A: Lepidium sativum, 3-d EC₅₀ = ca. 1000 mg/L (based on growth)

- Bioassay B: Brassica rapa, 10-d EC₅₀ = 52.1 mg/kg soil dw (based on growth; soil experiment)

- Bioassay C: Avena sativa, 8-wk EC₅ = ca. 3 mg/kg soil dw (based on growth)

The test design for bioassay (B) is considered most relevant as incubation conditions are similar to real world field conditions. Therefore, the 10-d EC₅₀ for Brassica rapa of 52.1 mg/kg soil dw is carried forward to the risk assessment.

Description of key information

The most sensitive reliable EC50 value for thiourea plant toxicity is reported to be 52.1 mg/kg soil dw(Günther & Pestemer, 1990). This value is carried forward to the risk assessment.

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:

52.1 mg/kg soil dw

Additional information

Two reliable studies (Günther & Pestemer, 1990 - key study; Pestemer & Auspurg, 1986) are available for the assessment of thiourea. In addition, two further studies with a Klimisch 3 rating are reported as supportive data.

Günther & Pestemer (1990) published results on the effects of thiourea on seedling germination and growth of the monocot Avena sativa (oats) and the dicots Brassica rapa (turnip) and Lepidium sativum (garden cress) that were studied in three different bioassays (A: 3-d growth on moistened filter paper; B: 10–14 days growth in soil and vermiculite; C: 8-wk growth in hydroponic culture (Avena sativa only)).

The most sensitive results in the different bioassays are as follows:

- Bioassay A: Lepidium sativum, 3-d EC₅ = ca. 100 mg/L

- Bioassay B: Brassica rapa, 10-d EC₅ = 21.1 mg/kg soil dw (soil experiment)

- Bioassay C: Avena sativa, 8-wk EC₅ = ca. 3 mg/kg soil dw

Respective EC₅₀ values (where applicable) are as follows:

Bioassay A: Lepidium sativum: EC₅₀ (3 d) = 1000 mg/L

Bioassay B: Brassica rapa (soil experiment): EC₅₀ (10d) = 52.1 mg/kg soil dw (soil experiment); Avena sativa (soil experiment): EC₅₀ (14 d) = 339.4 mg/kg soil dw

Pestemer and Auspurg (1986) investigated the effect of thiourea on seedling emergence and growth of the monocot Avena sativa and the dicots Brassica napus, Brassica rapa, Brassica campestris var. chinensis, and Lepidium sativum at nominal concentrations ranging between 0 and 1000 mg thiourea/kg soil dw in a 14-d study. The study design was similar to OECD 208 (1984). Depending on plant species, seed emergence and growth were affected by treatment with thiourea at concentrations of 100 mg/kg soil dw and/or 1000 mg/kg soil dw. The % inhibition in seed emergence and growth in the treated species as compared to the control ranged from ≤ 30 to ≥ 50 %. The only monocot species used in the test was Avena sativa with an EC₅₀ of < 1000 mg thiourea/kg soil dw.

For most dicot species no effects were observed at 1 mg/kg thiourea/kg soil dw (Brassica napus, Lepidium sativum), or even stimulation (Brassica rapa). Negative effects (≤ 30–50 % inhibition) could be observed at concentrations of 100 mg thiourea/kg soil dw. Therefore, in a worst case approach, the 14-d EC₅₀ is considered to be 100 mg thiourea/kg soil dw.

As the 10-d EC₅₀ value of 52.1 mg/kg soil dw for Brassica rapa determined by Günther & Pestemer (1990) is more sensitive than the 14-d EC₅₀ of 100 mg/k soil dw in the study by Pestemer & Auspurg (1986), the former is carried forward to the risk assessment.

Further supporting studies are available (Friesel et al., 1984; Korte and Freitag, 1984) which are, however, considered to be unreliable, and thus are disregarded in the hazard assessment (PNEC derivation).