2,6-di-tert-butyl-p-cresol

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 12th, 2017 to November 3rd, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 208 (Terrestrial Plants, Growth Test)

Version / remarks:

July 2006

Deviations:

yes

Remarks:

Due to technical reasons the number of replicates in treatment T5 of Brassica napus was six instead of seven. The deviation had no impact on the conduct or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 61.4 mg/mL
- Sampling method: the sample was left to stand to acclimatise to room temperature and homogenised by shaking directly before dilution. Two independent dilutions of each sample were prepared with acetonitrile in 100 mL volumetric flasks (dilution: 1:1000).
- Sample storage conditions before analysis: stored in a glass bottle at < -18°C (deep frozen)

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The test solutions were applied at a volume of 10 mL (“T1”) or 40 mL (“T2 to T6”) mL to 49.5 g or 162 g of quartz sand, respectively. The spiked sand was mixed thoroughly into 4.95 kg (“T1”) or 16.2 kg (“T2 to T6”) soil dry weight
equivalent by stirring for at least 5 minutes.
- Controls: The solvent control soil was mixed with the same amount of quartz sand that was treated with the solvent but without the test item. The control soil was mixed with the same amountof quartz sand that was neither treated with solvent nor with the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Evaporation of vehicle before use: Yes. The test item was dissolved in acetone and spiked onto quartz sand which was mixed thoroughly into the soil after complete evaporation of the solvent.

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Onion
- Plant family: Amaryllidaceae
- Source of seed: Enza Zaden
- Historical germination of seed (germination of seed lot tested): Stuttgarter Riesen, 4333216
- Seed storage: Stored under cool and dry conditions in the dark.

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Oat
- Plant family: Amaryllidaceae
- Source of seed: Nordsaat
- Historical germination of seed (germination of seed lot tested): Canyon, n.a.
- Seed storage: Stored under cool and dry conditions in the dark.

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Rape
- Plant family: Brassicaceae
- Source of seed: Feldsaaten
- Historical germination of seed (germination of seed lot tested): Akela, n.a.
- Seed storage: Stored under cool and dry conditions in the dark.

Species:

Pisum sativum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Cucumber
- Plant family: Cucurbitaceae
- Source of seed: Enza Zaden
- Historical germination of seed (germination of seed lot tested): Delikatess 4291508
- Seed storage: Stored under cool and dry conditions in the dark

Species:

Cucumis sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Pea
- Plant family: Fabaceae
- Source of seed: Enza Zaden
- Historical germination of seed (germination of seed lot tested): Rondo 117024NU
- Seed storage: Stored under cool and dry conditions in the dark

Species:

other: Solanum lycopersicum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Common name: Tomato
- Plant family: Solanaceae
- Source of seed: Enza Zaden
- Historical germination of seed (germination of seed lot tested): Hellfrucht 4292507
- Seed storage: Stored under cool and dry conditions in the dark

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

16 d

Remarks:

17 days for A. sativa, P. sativum; S. lycopersicum

Test temperature:

19.1-26.2 ºC

Moisture:

43.4%-64.6%

Details on test conditions:

TEST SYSTEM
- Testing facility: CIP – Chemisches Institut Pforzheim GmbH, Schulberg 17, 75175 Pforzheim, Germany
- Test container (type, material, size): plant pots made of polypropylene (diameter 11 cm, height 8.5 cm)
- Amount of soil: 480 ± 10 g
- No. of seeds per container: 7 (B. napus, C. sativus, P. sativum, S. lycopersicum) // 6 (A. cepa, A. sativa)
- No. of replicates per treatment group: 3 (B. napus, C. sativus, P. sativum, S. lycopersicum) // 4 (A. cepa, A. sativa)
- No. of replicates per control: 3 (B. napus, C. sativus, P. sativum, S. lycopersicum) // 4 (A. cepa, A. sativa)
- No. of replicates per vehicle control: 3 (B. napus, C. sativus, P. sativum, S. lycopersicum) // 4 (A. cepa, A. sativa)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Germany
- Pesticide use history at the collection site: he field had neither received any organic fertilisers nor plant protection substances during the last three years before sampling.
- Collection procedures: Sieving
- Sampling depth (cm): <2 mm
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- Organic carbon (%): 0.68 ± 0.04
- Maximum water holding capacity (in % dry weigth): 35.4 ± 1.0
- CEC: 7.5 ± 0.8 meq/100 g
- Storage (condition, duration): The soil was stored at ECT GmbH at ambient temperature until usage.

NUTRIENT MEDIUM (if used)
- Description: Substral®”, 7 ml/L H2O) was added twice within the first week after emergence (25 mL/pot and 50 mL/pot).

GROWTH CONDITIONS
- Photoperiod: 16 h
- Light source: SON-T-Agro high pressure metal halide lamps, Philips GmbH, Hamburg, Germany
- Light intensity and quality: 204-332 μE m-2 s-1
- Relative humidity (%): 43.4-64.6
- Watering regime and schedules: as needed
- Water source/type: deionised water


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Seedling Emergence and Seedling Survival: Day 0, 7 and end of the test
Visual appereance: day 7 and 14
Shoot Fresh Weight: end of the test

- Phytotoxicity rating system (if used):
1 Healthy Plant with normal growth, without damages apart from what iswithin the normal range of variability (e.g. wilted cotyledons are no
damage).
2 Slightly damaged Chlorotic and/or necrotic leaves (less than ¼ of the plant).
3 Slightly damaged Single wilted leaves (less than ¼ of the plant)
4 Severely damaged Chlorotic and/or necrotic and/or wilted leaves (more than ¼ of the
plant).
5 Severely damaged Plant with developmental abnormalities.
6 Dead All above-ground parts of the plant are wilted.


VEHICLE CONTROL PERFORMED: yes/

TEST CONCENTRATIONS
- Range finding study: A non-GLP range-finding test was performed in order to assess if the test item has adverse effects on the seedling emergence and growth of higher terrestrial plants at concentrations between 1.0 and 1000 mg/kg soil dry weight. (see table on additional information on materials and methods)
- Test concentrations: 1.00, 10.0,100,500, 1000 mg/kg soil dry weight.
- Results used to determine the conditions for the definitive study: Adverse effects on shoot fresh were observed for all test species at concentrations ≥100 mg/kg soil.

Nominal and measured concentrations:

Nominal concentrations: 61.4 mg/ml
Meeasured concentrations: 55 mg/ml

Reference substance (positive control):

no

Key result

Species:

Allium cepa

Duration:

16 d

Dose descriptor:

NOEC

Effect conc.:

4.74 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Key result

Species:

Allium cepa

Duration:

16 d

Dose descriptor:

LOEC

Effect conc.:

20.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

SEEDLING EMERGENCE
- Percent seedling emergence: A. cepa 95.5%, A. sativa 100%, B. napus 100%, C. sativus 95.0 %, P. sativum 100%, S. lycopersicum 100%
- Percent survival: A. cepa 91.7%, A. sativa 91.7%, B. napus 85.7%, C. sativus 95.2%, P. sativum 76.2%, S. lycopersicum 90.5%,
- Dry weight: see any other information on results
- Unusual leaf/plant shape or size: Plants growing in treated soil did not differ in their visual appearance from plants growing in non-treated control soil and did not exhibit any visible test item related damages.

INJURY RATING SYSTEM:
Rating score categories: 1 = no damages; 2/3 = slight damages; 4/5= severe damages; 6 = dead seedling; *Plants of pot “203” were not considered.

Reported statistics and error estimates:

Number of emerged and number of survived seedlings as well as shoot fresh weight at the end of the test were considered for statistical data analysis. Statistical methods were applied to evaluate whether differences between test item treatments and solvent control were significant. Arithmetic means for each pot were calculated from the single plants within a pot. Since each pot constituted a replicate, the mean values were used for statistical analysis. Plants which had emerged after day 7 were excluded from the calculations. Emergence and survival data were checked by the Fisher's exact binominal test with Bonferroni correction to determine the NOEC and LOEC. Significance level was alpha =0.05, one sided greater1. Probit, Weibull and Logit Analysis were used to determine the effective concentrations EC10 and EC50 for the end point shoot fresh weight. Normal distribution and homogeneity of variances were checked by the Kolmogorov-Smirnov Test and Cochran’s test, respectively. NOEC and LOEC of shoot fresh weight were determined by applying either the Williams multiple sequential t-test (monotonous doseresponse relationship). In case of inhomogeneous variances Welch t-Test was applied. Significance level was alpha = 0.05, one-sided smaller. Statistical evaluations were performed using the ToxRat Pro software Version 2.10 (ToxRat Solutions GmbH, 2010).

Table 2. Summary of the EC10, EC50 (with 95% confidence intervals, CI), LOEC, and NOEC of the endpoints emergence, survival and shoot fresh weight, referring to nominal test item concentration [mg/kg soil dry weight].

Species	NOEC	LOEC	EC10	EC50
Emergence
A. cepa	≥150	>150	n.d.	n.d.
A. sativa	47.4	150	n.d.	n.d.
B. napus	≥150	>150	n.d.	n.d.
C. sativus	≥150	>150	n.d.	n.d.
P. sativum	47.4	150	n.d.	n.d.
S. lycopersicum	≥150	>150	n.d.	n.d.
Survival
A. cepa	47.4	150	48.0 (17.5 - 70.5)	128 (90.2 - 234)
A. sativa	≥150	>150	n.d.	n.d.
B. napus	≥150	>150	n.d.	n.d.
C. sativus	≥150	>150	n.d.	n.d.
P. sativum	≥150	>150	n.d.	n.d.
S. lycopersicum	≥150	>150	n.d.	n.d.
Shoot fresh weight
A. cepa	4.74	15.0	8.1 (5.0 - 10.3)	20.9 (17.8 - 25.9)
A. sativa	47.4	150	n.d.	n.d.
B. napus	47.4	150	47.7 (30.0 - 61.6)	90.9 (73.0 - 109)
C. sativus	47.4	150	n.d.	n.d.
P. sativum	47.4	150	31.3 (30.8 - 31.7)	57.6 (57.1 - 58.0)
S. lycopersicum	15.0	47.4	29.3 (0.0 - 61.7)	124 (53.0 - 7884)

n.d.: not determined / calculation not feasible (e.g. no dose-response relationship).

Table 3. Number of seeds introduced, number of emerged seeds, and number of survived seedlings in the untreated control pots.

Plant species	Seeds	Day 0	Emerged	Survived	Emerged	Survived
	[No.]1	[d] 2	[No.] 3	[No.] 4	[%]	[%]
A. cepa	24	5	22	21	91.7	95.5
A. sativa	24	4	22	22	91.7	100
B. napus	21	4	18	18	85.7	100
C. sativus	21	4	20	19	95.2	95.0
P. sativum	21	5	16	16	76.2	100
S. lycopersicum	21	5	19	19	90.5	100

Table 4. Seedling emergence

Test item		A. cepa		A. sativa		B. napus			C. sativus	P. sativum			S.lycopersicum
Number of seeds planted
C	0.0		24		24		21	21			21	21
SC	0.0		24		24		21	21			21	21
T1	0.47		24		---		21	---			---	---
T2	1.5		24		24		21	21			21	21
T3	4.74		24		24		21	21			21	21
T4	15.0		24		24		21	21			21	21
T5	47.74		24		24		21	21			21	21
T6	150		24		24		21	21			21	21
Number of emerged seeds per treatment on day 7a
C	0.0		22		22		18	20			16	19
SC	0.0		20		23		19	16			18	20
T1	0.47		20		---		18	---			---	---
T2	1.5		21		23		20	18			15	19
T3	4.74		21		22		19	18			17	19
T4	15.0		19		24		19	16			18	20
T5	47.74		19		20		16	17			16	20
T6	150		19		9		18	17*			8*	20
Relative number of emerged seeds [% of seeds sown]
C	0.0		91.7		91.7		85.7	95.2			76.2	90.5
SC	0.0		83.3		95.8		90.5	76.2			85.7	95.2
T1	0.47		83.3		---		85.7	---			---	---
T2	1.5		87.5		95.8		95.2	85.7			71.4	90.5
T3	4.74		87.5		91.7		90.5	85.7			81.0	90.5
T4	15.0		79.2		100		90.5	76.2			85.7	95.2
T5	47.74		79.2		83.3		88.9	81.0			76.2	95.2
T6	150		79.2		37.5*		85.7	81.0			38.1*	95.2
Number of live seedlings at test end
C	0.0		21		22		18	19			16	19
SC	0.0		19		23		19	16			17	20
T1	0.47		20		---		18	---			---	---
T2	1.5		21		23		20	18			15	19
T3	4.74		21		21		19	18			17	19
T4	15.0		20		24		19	16			18	20
T5	47.74		17		20		16b	16			16	20
T6	150		8*		8		18	17			8	20
Relative number of live seedlings at test end [% of emerged]
C	0.0		95.5		100		100	95.0			100	100
SC	0.0		95.0		100		100	100			94.4	100
T1	0.47		100		---		100	---			---	---
T2	1.5		100		100		100	100			100	100
T3	4.74		100		95.5		100	100			100	100
T4	15.0		100		100		100	100			100	100
T5	47.74		89.5		100		100	94.1			100	100
T6	150		42.1*		88.9		100	100			100	100

(---) = respective treatment not tested; (a) = post day 7 emergence not considered; (b) = pot no 203 not considered; * = statistically significantly different from the solvent control (p<0.05, one-sided greater).

Table 5. Visual appearance of seedlings on day 7 according to a 6 point scoring system (post day 7 emergence not considered).

Species	Code	Test item [mg/kg soil dw]	1	2	3	4	5	6
A. cepa	C	0.0	22	---	---	---	---	---
	SC	0.0	19	---	---	1	---	---
	T1	0.47	20	---	---	---	---	---
	T2	1.50	21	---	---	---	---	---
	T3	4.74	21	---	---	---	---	---
	T4	15.0	19	---	---	---	---	---
	T5	47.4	19	---	---	---	---	---
	T6	150	18	---	---	1	---	---
A. sativa	C	0.0	22	---	---	---	---	---
	SC	0.0	23	---	---	---	---	---
	T2	1.50	23	---	---	---	---	---
	T3	4.74	22	---	---	---	---	---
	T4	15.0	24	---	---	---	---	---
	T5	47.4	20	---	---	---	---	---
	T6	150	---	7	---	---	2	---
B. napus	C	0.0	18	---	---	---	---	---
	SC	0.0	19	---	---	---	---	---
	T1	0.47	18	---	---	---	---	---
	T2	1.50	20	---	---	---	---	---
	T3	4.74	19	---	---	---	---	---
	T4	15.0	19	---	---	---	---	---
	T5	47.4	16	---	---	---	---	1
	T6	150	---	18	---	---	---	---
C. sativus	C	0.0	19	---	---	---	---	1
	SC	0.0	16	---	---	---	---	---
	T2	1.50	17	1	---	---	---	---
	T3	4.74	17	1	---	---	---	---
	T4	15.0	15	1	---	---	---	---
	T5	47.4	16	1	---	---	---	---
	T6	150	---	17	---	---	---	---
P. sativum	C	0.0	16	---	---	---	---	---
	SC	0.0	17	---	---	---	1	---
	T2	1.50	15	---	---	---	---	---
	T3	4.74	17	---	---	---	---	---
	T4	15.0	18	---	---	---	---	---
	T5	47.4	---	16	---	---	---	---
	T6	150	---	---	---	---	8	---
S.lycopersicum	C	0.0	19	---	---	---	---	---
	SC	0.0	20	---	---	---	---	---
	T2	1.50	19	---	---	---	---	---
	T3	4.74	19	---	---	---	---	---
	T4	15.0	20	---	---	---	---	---
	T5	47.4	20	---	---	---	---	---
	T6	150	20	---	---	---	---	---

Rating score categories: 1 = no damages; 2/3 = slight damages; 4/5= severe damages; 6 = dead seedling

Table 6. Visual appearance of seedlings at the end of the test according to a 6 point scoring system (post day 7 emergence not considered).

Species	Code	Test item [mg/kg soil dw]	1	2	3	4	5	6
A. cepa	C	0.0	21	---	---	---	---	1
	SC	0.0	19	---	---	---	---	1
	T1	0.47	19	1	---	---	---	---
	T2	1.50	19	1	1	---	---	---
	T3	4.74	20	1	---	---	---	---
	T4	15.0	19	---	---	---	---	1
	T5	47.4	---	12	---	5	---	2
	T6	150	---	3	---	5	---	11
A. sativa	C	0.0	22	---	---	---	---	---
	SC	0.0	23	---	---	---	---	---
	T2	1.50	23	---	---	---	---	---
	T3	4.74	21	---	---	---	---	1
	T4	15.0	24	---	---	---	---	---
	T5	47.4	20	---	---	---	---	---
	T6	150	3	5	---	---	---	1
B. napus	C	0.0	18	---	---	---	---	---
	SC	0.0	19	---	---	---	---	---
	T1	0.47	18	---	---	---	---	---
	T2	1.50	20	---	---	---	---	---
	T3	4.74	19	---	---	---	---	---
	T4	15.0	19	---	---	---	---	---
	T5*	47.4	16	---	---	---	---	1
	T6	150	3	11	4	---	---	---
C. sativus	C	0.0	19	---	---	---	---	1
	SC	0.0	16	---	---	---	---	---
	T2	1.50	17	1	---	---	---	---
	T3	4.74	17	---	1	---	---	---
	T4	15.0	14	2	---	---	---	---
	T5	47.4	13	1	2	---	---	1
	T6	150	16	1	---	---	---	1
P. sativum	C	0.0	12	3	1	---	---	---
	SC	0.0	10	7	---	---	---	1
	T2	1.50	12	3	---	---	---	---
	T3	4.74	9	8	---	---	---	---
	T4	15.0	13	5	---	---	---	---
	T5	47.4	15	1	---	---	---	---
	T6	150	8	---	---	1	---	---
S.lycopersicum	C	0.0	19	---	---	---	---	---
	SC	0.0	20	---	---	---	---	---
	T2	1.50	19	---	---	---	---	---
	T3	4.74	19	---	---	---	---	---
	T4	15.0	19	1	---	---	---	---
	T5	47.4	19	---	1	---	---	---
	T6	150	19	1	---	---	---	---

Rating score categories: 1 = no damages; 2/3 = slight damages; 4/5= severe damages; 6 = dead seedling

Table 7. Mean shoot fresh weight at the end of the test; absolute [g] and as [%] related to the solvent control.

Treatment code	C	SC	T1	T2	T3	T4	T5	T6
Test item [mg/kg soil dw]	0.0	0.0	0.47	1.5	4.74	15.0	47.4	150
Allium cepa
Mean [g]	0.440	0.412	0.450	0.394	0.430	0.285*	0.016*	0.013*
SD	0.042	0.018	0.044	0.077	0.017	0.031	0.007	0.003
% reduction a	n.d.	n.a.	-9.2	4.4	-4.1	30.9	96.1	96.9
Avena sativa
Mean [g]	2.084	1.930	---	2.022	2.070	2.036	2.090	0.435*
SD	0.109	0.093	---	0.294	0.260	0.130	0.060	0.481
% reduction a	n.d.	n.a.	---	-4.7	-7.2	-5.5	-8.3	77.4
Brassica napus
Mean [g]	4.087	3.789	3.944	3.534	3.767	3.701	3.421	0.582*
SD	0.726	0.739	0.650	0.355	0.789	0.664	0.641	0.168
% reduction a	n.d.	n.a.	-4.1	6.7	0.6	2.3	9.7	84.6
Cucumis sativus
Mean [g]	5.314	6.367	---	4.870	5.074	5.216	3.740	2.438*
SD	1.511	2.495	---	0.708	1.130	0.710	0.944	0.309
% reduction a	n.d.	n.a.	---	23.5	20.3	18.1	41.3	61.7
Pisum sativum
Mean [g]	3.891	2.701	---	3.490	3.321	2.772	1.778	0.060*
SD	1.206	0.347	---	0.647	0.571	0.410	0.962	0.018
% reduction a	n.d.	n.a.	---	-29.2	-23.0	-2.6	34.2	97.8
S. lycopersicum
Mean [g]	3.831	3.705	---	3.219	3.618	3.515	3.055	1.571
SD	0.663	0.529	---	0.730	0.340	0.612	0.288	0.454
% reduction a	n.d.	n.a.	---	13.1	2.3	5.1	17.5	57.6

C = control; SC = solvent control; T1 to T6 = test item treated; a = compared to solvent control; (---) = not tested; n.a. = not applicable; n.d. = not determined; * = significantly different from solvent control (p=0.05, one sided smaller).

Validity criteria fulfilled:

yes

Remarks:

The validity criteria were fulfilled as seedling emergence in the control was ≥ 76.2% and seedling survival in the control was ≥ 95.0% for all six species. No phytotoxicity was observed in the controls.

Conclusions:

The overall lowest NOEC and EC50 for the test item were 4.74 mg/kg soil dry weight and 20.9 mg/kg soil dry weight, respectively, and were observed with shoot fresh weight of Allium cepa.

Executive summary:

A long term toxicity test to terrestrial plants was performed following OECD 208 and under GLP conditions. Seeds of two monocotyledonous species, Allium cepa (onion) and Avena sativa (oat), as well as four dicotyledonous species, Brassica napus (rape), Cucumis sativus (cucumber), Pisum sativum (pea) and Solanum lycopersicum (tomato) were planted in a natural sandy loam soil (standard soil LUFA Sp 2.3) immediately after test item application and left to grow under controlled conditions for either 16 days and concentrations ranging from 0.47 to 150 mg/kg soil dry weight (monocotyledonous species) or 17 days and concentrations ranging from 1.50 to 150 mg/kg soil dw (dicotyledonous species) , following 50 % emergence of the control plants. In all species, a spacing factor of 3.2 was used. A sample of the highest treatment solution was taken to verify the test concentration by chemical analysis using a validated GC method. Soil for growing control plants was amended either with non-spiked sand (control) or with acetone spiked sand (solvent control). The test was performed in a growth chamber. On day 7 after 50%, the emergence of control seedlings was evaluated visually. At the end of the test, (day 16 or 17), seedlings were counted, evaluated visually, and harvested to determine shoot fresh weight. All validity criteria were met. The overall lowest NOEC and EC50 for the test item were 4.74 mg/kg soil dry weight and 20.9 mg/kg soil dry weight, respectively, and were observed with shoot fresh weight of Allium cepa.

Description of key information

Key study. Method according to OECD 208, GLP study. The lowest EC50 based on shoot fresh weight was 20.9 mg/kg soil dry weight for Alium cepa.

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:

20.9 mg/kg soil dw

Additional information

A long term toxicity test to terrestril plants was performed following OECD 208 and under GLP conditions. Seeds of two monocotyledonous species,Allium cepa(onion) andAvena sativa(oat), as well as four dicotyledonous species,Brassica napus(rape),Cucumis sativus(cucumber),Pisum sativum(pea) andSolanum lycopersicum(tomato) were planted in a natural sandy loam soil (standard soil LUFA Sp 2.3) immediately after test item application and left to grow under controlled conditions for either 16 days and concetrations ranging from 0.47 to 150 mg/kg soild dry weight (monocotyledonous species) or 17 days and concetrations ranging from 1.50 to 150 mg/kg soil dw (dicotyledonous species) , following 50 % emergence of the control plants. On day 7 after 50% emergence of control seedlings were evaluated visually. At the end of the test, (day 16 or 17), seedlings were counted, evaluated visually, and harvested to determine shoot fresh weight. All validity criteria were met. The overall lowest NOEC and EC50 for the test item were 4.74 mg/kg soil dry weight and 20.9 mg/kg soil dry weight, respectively, and were observed with shoot fresh weight of Allium cepa.