REAKTIV-GELB F-68 072 FW

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:

supporting study

Study period:

01 June 2006 to 21 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

yes

Details on sampling:

Five different test item concentrations (T1 - T5), and respective controls (TO).
Nominal concentrations of the test item in soil were 0.0 mg a.s./kg (TO, control), 12.3 mg a.s./kg (T1), 37.0 mg a.s./kg (T2), 111.1 mg a.s./kg (T3), 333.3 mg a.s./kg (T4) and 1000 mg a.s./kg (T5).

A 200 mL sample of the stock solution was always taken immediately prior to mixing the test item into the soil on June 23, 2006 (test with A. sativa), and on June 30, 2006 (test with B. napus and L. lycopersicon), respectively, and stored at minus 18°C, or below, until chemical analysis

Vehicle:

no

Details on preparation and application of test substrate:

The test item was miscible with water, it was dissolved in demineralised water and mixed into the soil immediately prior to sowing of the seeds.
Five different test item concentrations (T1 - T5), and respective controls (TO) were tested with each of the three plant species. Controls were treated with water instead of the test item solution. Nominal concentrations of the test item in soil were 0.0 mg a.s./kg (TO, control), 12.3 mg a.s./kg (T1), 37.0 mg a.s./kg (T2), 111.1 mg a.s./kg (T3), 333.3 mg a.s./kg (T4) and 1000 mg a.s./kg (T5). There were 4 replicates (pots) with 6 plant specimens each, for every test item concentration, and for the control, in the case of A.sativa, and 5 replicates (pots) with four plant specimens each, for every test item concentration, and for the control, in the case of B. napus and L. lycopersicon.

Standard soil LUFA Type 2.3, a sandy loam was purchased from the "Landwirtschaftliche Untersuchungs- und Forschungsanstalt", D-67346 Speyer. Information on texture, organic carbon content and pH was provided by the supplier of the soil. The actual water content of the soil was determined before the test.

A stock solution was prepared by dissolving 39.209 g of the test item in demineralised water at a total volume of 2.0 L. Further dosage solutions were obtained by filling up defined volumes of the stock solution with demineralised water to a total volume of 1.10 L. To achieve the required test item concentrations in the soil, volumes of 1.10 L of the stock solution, and of the respective dosage solutions, or water (control), were mixed into soil quantities of 10.5668 kg (dry weight equivalent), each.

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Plant Species: Avena sativa (oat)
Type: Aragon
Source: Nordsaat Saatzuchtgesellschaft m.b.H.

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Plant Species: Brassica napus, var. oleifera subvar, biennis (oilseed rape)
Type: Akela
Source: LUFA Speyer

Species:

Lycopersicon esculentum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Plant Species: Lycopersicon lycopersicum (tomato)
Type: Hellfrucht/Selektion JW
Source: Juliwa Markensaat

Test type:

seedling emergence toxicity test

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

No post exposure observation period

Test temperature:

24 ± 2°C

pH:

No data

Moisture:

No data

Details on test conditions:

Application of the Test Item
A stock solution was prepared by dissolving 39.209 g of the test item in demineralised water at a total volume of 2.0 L. Further dosage solutions were obtained by filling up defined volumes of the stock solution with demineralised water to a total volume of 1.10 L. To achieve the required test item concentrations in the soil, volumes of 1.10 L of the stock solution, and of the respective dosage solutions, or water (control), were mixed into soil quantities of 10.5668 kg (dry weight equivalent), each.

Test Containers
Rectangular polystyrene beakers, 18.3 cm by 13.6 cm large and 6.4 cm high (type 590, Kastelplast GmbH, Mainz-Kastel, Germany) were used as test containers (pots). A glass fibre wick of 10 mm diameter (Isoglas-Packung, Frenzelit Werke GmbH, Bad Berneck, Germany) was inserted through a 10 mm hole punched into the bottom of the beaker. The pots were placed above a second polystyrene beaker serving as a water reservoir in a way that the wicks reached the water reservoirs. Each pot was filled with 800 ± 10 g of either treated or non-treated (control) LUFA Sp2.3 standard soil. The soil surface was smoothened carefully, avoiding compression of the soil.

Sowing
Seeds of the plant species were sown either into the untreated control soil, or into the soil treated with the test item. The seeds have neither been dressed with fungicides nor imbibed with water.
The seeds were randomly picked with a pair of tweezers and pressed 5-10 mm into the soil at a uniform distance between seeds and at least 5 mm away from the rim of the pot. The soil surface was carefully smoothened afterwards. Each pot of Avena sativa received six seeds, while each pot of Brassica napus and Lycopersicon lycopersicum received 4 seeds, respectively.
The pots were labelled individually with the study number and with a consecutive number indicating the plant species and the treatment according to a numbering scheme Randomisation
Random numbers for all pots of each test species were generated using a function provided by Microsoft Excel. According to these numbers, three to four of the pots were placed on trays. Thereafter, the trays were distributed randomly in the test room according to a second set of random numbers. Re-randomisation was done by weekly re-arranging the trays.

Light and Temperature
The test plants were cultivated in a room equipped with artificial lighting (SON-T-Agro high pressure metal halide lamps, Philips GmbH, Hamburg, Germany) with a photoperiod of 16 h/d. The average light intensity measured at the soil surface across the test area was 12888 ± 1239 Ix (mean and standard deviation, n=30). The temperature in the test rooms was kept in the range of 24 ± 2°C. Light and temperature were controlled and documented in the area where the trays were placed.

Irrigation
After placing the pots in the test room, their water reservoirs were filled with a volume of deionised water sufficient to moisten the soil. During the test, water was added to the reservoirs as necessary. When the plants had produced their cotyledons or reached the one-leaf stage the reservoirs were re-filled with a nutrient solution (a dilution of a commercially available liquid fertilizer).

Nominal and measured concentrations:

Nominal concentrations of the test item in soil were 0.0 mg a.s./kg (TO, control), 12.3 mg a.s./kg (T1), 37.0 mg a.s./kg (T2), 111.1 mg a.s./kg (T3), 333.3 mg a.s./kg (T4) and 1000 mg a.s./kg (T5).

Reference substance (positive control):

no

Species:

Avena sativa

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks on result:

other: Additional results for survival, shoot length and fresh weight are tabulated below.

Species:

Avena sativa

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks on result:

other: Additional results for survival, shoot length and fresh weight are tabulated below.

Species:

Brassica napus

Duration:

21 d

Dose descriptor:

LC25

Effect conc.:

155.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks on result:

other: Additional results for survival, shoot length and fresh weight are tabulated below.

Species:

Brassica napus

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

4 978 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks on result:

other: Additional results for survival, shoot length and fresh weight are tabulated below.

Species:

Brassica napus

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

333.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks on result:

other: Additional results for survival, shoot length and fresh weight are tabulated below.

Species:

Brassica napus

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks on result:

other: Additional results for survival, shoot length and fresh weight are tabulated below.

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks on result:

other: Additional results for survival, shoot length and fresh weight are tabulated below.

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks on result:

other: Additional results for survival, shoot length and fresh weight are tabulated below.

Details on results:

LC50 values for seedling emergence could only be calculated for B. napus (4978 mg). LC50 values for seedling survival could only be calculated for B. napus (1145 mg) and L. Lycopersicon (1057 mg). The lowest LC25 values for emergence and survival were 155.9 mg and 200.4 mg a.i./kg soil dry weight, respectively.
The lowest EC50 values for seedling shoot length, and seedling shoot mass were 714.8 mg, and 371.2 mg a.i./kg soil dry weight, respectively.
The test item had no effect on seedling emergence and growth of higher plants at concentrations up to 111.1 mg a.i./kg soil dry mass.

Further details in table form are listed below under any other information

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The Probit Analysis was used to determine LC50, LC25, EC50 and EC25. Normal distribution was checked by the RS-Test, and homogeneity of the variances by Cochran's Test Procedure. NOEC and LOEC were determined by applying the Williams Test for homogeneous variances, and by Welch-t Test for inhomogeneous variances. In case the data did not follow normal distribution a Bonferroni-Holm U-Test was performed. Significance level was always Alpha = 0.05.
All statistical evaluations were performed using the ToxRat software Version 2.09 (ToxRat Solutions GmbH, 2006).

As more than 70% of the controls emerged, less than 10% of the control plants died, and most control plants developed healthily, the quality criteria defined in the draft test guideline and the study plan have been fulfilled.

Seedling emergence in the control pots.

	Avena sativa	Brassica napus	Lycopersicon lycopersium
Number of seeds sown	24	20	20
Date of Day 0 of the test  [days after sowing]	June 27, 2006 [4]	July 04, 2006 [4]	July 05, 2006 [5]
Number of emerged seedlings	24	18	18
Number of seedlings [% of sown]	100	90.0	90.0

 

Number of emerged seedlings within the test period

Test Item  [mg a.i./kg dry soil]	Avena sativa		Brassica napus		Lycopersicon lycopersicum
	No.	%	No.	%	No.	%
(control) 0.0	24	100.0	18	90.0	19	95.0
12.3	24	100.0	17	85.0	19	95.0
37.0	24	100.0	17	85.0	15	75.0
111.1	24	100.0	15	75.0	16	80.0
333.3	24	100.0	16	80.0	15	75.0
1000.0	23	95.8	11	55.0	19	95.0

 

Number of seedlings at the end of the test in % of the number if emerged seedlings

Test item  [mg a.i./kg dry soil]	Avena sativa	Brassica napus	Lycopersicon lycopersicum
(control) 0.0	100	100	94.7
12.3	100	100	94.7
37.0	100	100	93.3
111.1	100	100	81.3
333.3	100	93.8	73.3
1000.0	100	54.5	47.4

 

Summary of the EC/LC₂₅, EC/LC₅₀, NOEC and LOEC of the endpoints emergence, survival, shoot length and shot fresh weight.

Species	A. sativa	B. napus	L. lycopersicum
Emergence
LC25	n.d.	155.9	n.d.
LC50	n.d.	4978	n.d.
NOEC	≥ 1000	333.3	≥ 1000
LOEC	> 1000	1000	> 1000
Survival
LC25	n.d.	645.8	200.4
LC50	n.d.	1144.6	1056.9
NOEC	≥ 1000	333.3	333.3
LOEC	> 1000	1000	1000
Shoot length
EC25	n.d.	359	296.4
EC50	n.d.	714.8	1083.8
NOEC	≥ 1000	333.3	111.1
LOEC	> 1000	1000	333.3
Fresh weight
EC25	967.5	185	116.6
EC50	1300.8	371.2	289.8
NOEC	333.3	111.1	111.1
LOEC	1000	333.3	333.3

n.d. not determined/calculation not feasible due to the results.

Validity criteria fulfilled:

yes

Conclusions:

The test item had no effect on seedling emergence and growth of higher plants at concentrations up to 111.1 mg a.i./kg soil dry mass.

Executive summary:

Study conducted to OECD Guideline for the Testing of Chemicals; Proposal for Updating Guideline 208 (Draft Document March 2005); Terrestrial Plant Test: 208: Seedling Emergence and Seedling Growth Test, in compliance with the Principles of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

Seeds of one monocotyledoneous species(Avena sativa)and two dicotyledoneous species(Brassica napus, Lycopersicon lycopersicum)were planted in a standard soil, LUFA Sp2.3. Immediately before sowing, the test item was dissolved in deionised water and mixed into the soil at 5 concentrations (dose response test) ranging from 12.3 mg a.i./kg to 1000 mg a.i./kg soil dry weight. Controls were treated with water only. Following treatment, the plants were allowed to emerge and to grow for up to 21 days following 50% emergence of the control plants under laboratory conditions. Soils were supplied with water or nutrient solution by glass fibre wicks. The test was performed in a growth chamber at a temperature of 24 ± 2°C and lighting of 13000 ± 2000 Ix (16 hours per day). The pots were placed randomly at the beginning of the test and were re-arranged several times during the incubation period. At day 7 and 14 after 50% of the control seedling had emerged, a visual inspection was done. On day 17(B. napus),day 16(L. Lycopersicon)and on day 21(A. sativa)the plants were counted, visually inspected to detect possible damages, and harvested to determine shoot length and shoot fresh weight.

 

The lowest NOEC of the test item for the endpoints seedling emergence, seedling survival, seedling shoot length, and seedling shoot fresh weight of the three tested species of higher plants were 333.3 mg, 333.3 mg, 111.1 mg, and 111.1 mg a.i./kg soil dry weight, respectively.

 

LC50 values for seedling emergence could only be calculated forB. napus(4978 mg). LC50 values for seedling survival could only be calculated forB. napus(1145 mg) andL. Lycopersicon(1057 mg). The lowest LC25 values for emergence and survival were 155.9 mg and 200.4 mg a.i./kg soil dry weight, respectively.

 

The lowest EC50 values for seedling shoot length, and seedling shoot mass were 714.8 mg, and 371.2 mg a.i./kg soil dry weight, respectively.

 

The test item had no effect on seedling emergence and growth of higher plants at concentrations up to 111.1 mg a.i./kg soil dry mass.

Description of key information

The test item had no effect on seedling emergence and growth of higher plants at concentrations up to 111.1 mg a.i./kg soil dry mass.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:

111.1 mg/kg soil dw

Additional information

Study conducted to OECD Guideline for the Testing of Chemicals; Proposal for Updating Guideline 208 (Draft Document March 2005); Terrestrial Plant Test: 208: Seedling Emergence and Seedling Growth Test, in compliance with the Principles of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

Seeds of one monocotyledoneous species(Avena sativa)and two dicotyledoneous species(Brassica napus, Lycopersicon lycopersicum)were planted in a standard soil, LUFA Sp2.3. Immediately before sowing, the test item was dissolved in deionised water and mixed into the soil at 5 concentrations (dose response test) ranging from 12.3 mg a.i./kg to 1000 mg a.i./kg soil dry weight. Controls were treated with water only. Following treatment, the plants were allowed to emerge and to grow for up to 21 days following 50% emergence of the control plants under laboratory conditions. Soils were supplied with water or nutrient solution by glass fibre wicks. The test was performed in a growth chamber at a temperature of 24 ± 2°C and lighting of 13000 ± 2000 Ix (16 hours per day). The pots were placed randomly at the beginning of the test and were re-arranged several times during the incubation period. At day 7 and 14 after 50% of the control seedling had emerged, a visual inspection was done. On day 17(B. napus),day 16(L. Lycopersicon)and on day 21(A. sativa)the plants were counted, visually inspected to detect possible damages, and harvested to determine shoot length and shoot fresh weight.

 

The lowest NOEC of the test item for the endpoints seedling emergence, seedling survival, seedling shoot length, and seedling shoot fresh weight of the three tested species of higher plants were 333.3 mg, 333.3 mg, 111.1 mg, and 111.1 mg a.i./kg soil dry weight, respectively.

 

LC50 values for seedling emergence could only be calculated forB. napus(4978 mg). LC50 values for seedling survival could only be calculated forB. napus(1145 mg) andL. Lycopersicon(1057 mg). The lowest LC25 values for emergence and survival were 155.9 mg and 200.4 mg a.i./kg soil dry weight, respectively.

 

The lowest EC50 values for seedling shoot length, and seedling shoot mass were 714.8 mg, and 371.2 mg a.i./kg soil dry weight, respectively.