1,2,3-Propanetricarboxylic acid, 2-(1-oxobutoxy)-, 1,2,3-trihexyl ester

Toxicity to terrestrial plants

Administrative data

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Test Guideline 208

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

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)

Remarks:

OECD GLP; UK GLP; EC Commission Directive 2004/10/EC

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

413-890-4

EC Name:

-

Cas Number:

82469-79-2

Molecular formula:

C28H50O8

IUPAC Name:

1,2,3-trihexyl 2-(butanoyloxy)propane-1,2,3-tricarboxylate

Constituent 2

Reference substance name:

1,2,3-propanetricarboxylic acid, 2-(butoxy), tri-n-hexyl est er

IUPAC Name:

1,2,3-propanetricarboxylic acid, 2-(butoxy), tri-n-hexyl est er

Details on test material:

Citroflex® B-6; n-Butyryltri-n-hexyl Citrate (BTHC); CAS # 82469-79-2; Lot/Batch No. 0000085635; Expiration Date 06 July 2009; Purity, 99.8%; Clear oily liquid; Storage conditions, room temperature in the dark.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Test concentrations were corrected for the purity of the test substance.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

On the day of preparation, aliquots (15 mL) of the treatment solutions prepared for highest treatment level for each species were collected for verification of Citroflex® B-6 concentration.

Test substrate

Vehicle:

yes

Details on preparation and application of test substrate:

The soil used was a loamy sand (classification determined prior to amendment with sand during preparation for treatment). The carbon content, pH and particle size of the soil were analysed and it was sieved prior to use to remove all particles >2 mm. The percentage organic matter was 1.38%, by calculation, based on the measured organic carbon percentage of 0.8%. The pH was 6.5 and the particle size distribution was as follows (% by weight): sand, 89%; silt, 2%; clay, 9%

Test substance treatment solutions were prepared in acetone and were poured over premeasured aliquots of sand. For the solvent control groups, acetone only was added to the sand. When the acetone had evaporated (approximately 2 to 4 hours), the sand was mixed into the appropriate amount of weighed moist soil. For the untreated control groups, only sand was added to the soil. Once the sand had been mixed into the soil, the soil was moistened with reverse osmosis (RO) water, mixed using a handheld mixer, and distributed among labelled replicate pots.

Test organisms

1

Species:

Brassica oleracea var. capitata

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Seeds were purchased from a commercial supplier and were stored dry in glass bottles in darkness prior to the start of the study. A germination trial demonstrated that seed viability was > 80% Mean weight per seed was 0.00107 g.

2

Species:

other: Vigna radiata (mung bean)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Seeds were purchased from a commercial supplier and were stored dry in glass bottles in darkness prior to the start of the study. A germination trial demonstrated that seed viability was > 80% Mean weight per seed was 0.0717 g.

3

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Seeds were purchased from a commercial supplier and were stored dry in glass bottles in darkness prior to the start of the study. A germination trial demonstrated that seed viability was > 80% Mean weight per seed was 0.0491 g.

Study design

Test type:

other: Seedling emergence and growth

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

2 wk

Remarks:

14 to 15 days following 50% germination in the control groups.

Post exposure observation period:

None

Test conditions

Test temperature:

Cabbage: 19.9 to 23.6°C
Mung bean: 24.6 to 27.1°C
Wheat: 18.0 to 21.4°C

pH:

Cabbage: 6.7 to 7.1
Mung bean: 6.8 to 7.2
Wheat: 6.9 to 7.4

Moisture:

2.3% prior to treatment.

Details on test conditions:

A preliminary range finding test was carried out at test concentrations of 0.1, 1, 10, 100 and 1000 mg a.i/Kg dry soil. Fifteen seeds were sown with three seeds per pot for cabbage and wheat for all treatment levels. Sixteen seeds with two seeds per pot were sown for mung bean for all treatment levels. The test included untreated water control and solvent control groups. Based on the results of the range finder, definitive treatment levels of 62.5, 125, 250, 500 and 1000 mg a.i./Kg dry soil were selected for wheat and a single definitive treatment level (i.e. limit test) of 1000 mg a.i/Kg dry soil was selected for cabbage and mung bean.

For the definitive study, a total of 14 replicates, each containing three seeds were used for cabbage and wheat, 20 replicate pots of two seeds each were used for mung bean. For all species 10 cm internal diameter plastic pots were used and each was filled to a depth of approximately 75mm. The seeds were spaced out on the soil surface and approximately 3 mm of the same soil was added to cover the seeds. Each pot was labelled with the study number, species name, dose group, replicate number and Study Director. All pots were situated under lights (scheduled to give 16 hours of continuous light per day) in suitable environmentally controlled rooms. Plants were top watered daily until germination and then top and/or bottom watered with RO water as required to maintain seedling health and growth (visually determined on a per-species and per-treatment basis). Temperatures were maintained at levels suitable for the individual species. Temperature, humidity and light intensity, (initially at soil level then at the top of the canopy after germination) were recorded daily throughout the exposure period.

Each pot was inspected daily and the number of emerged plants was recorded, together with any herbicidal symptoms. Emergence was defined as any plant material observed above the soil surface. After 50% emergence in the controls was achieved, observations continued every two to three days until the plants were harvested. Observations included visual assessments based on the overall condition of surviving seedlings. These assessments were conducted using a uniform scoring system based on plant size and vigour, where 0 indicated no abnormal effects and 10 indicated plant death. Plants from all treatment and control groups were harvested 14 - 15 days after germination of at least 50% of the control seedlings, by cutting each stem at soil level. The wet weight of each plant was then recorded before they were placed into individually labelled paper bags and dried to a constant weight at approximately 60ºC at which time the dry weight was recorded.

Nominal and measured concentrations:

Cabbage and mung bean: 0 mg a.i./kg (water and solvent control), 1000 mg a.i/kg dry soil (nominal)
Wheat: 0 mg a.i./kg (water and solvent control), 62.5, 125, 250, 500 and 1000 mg a.i./kg dry soil (nominal)

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Species:

Brassica oleracea var. capitata

Duration:

2 wk

Dose descriptor:

LC50

Effect conc.:

> 1 000 other: mg a.i./kg soil d.w.

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: germination and survival

Effect concentrations 2

Species:

Brassica oleracea var. capitata

Duration:

2 wk

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 other: mg a.i./kg soil d.w.

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: germination and survival

Effect concentrations 3

Species:

other: Vigna radiata (mung bean)

Duration:

2 wk

Dose descriptor:

LC50

Effect conc.:

> 1 000 other: mg a.i./kg soil d.w.

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: germination and survival

Effect concentrations 4

Species:

other: Vigna radiata (mung bean)

Duration:

2 wk

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 other: mg a.i./kg soil d.w.

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: germination and survival

Effect concentrations 5

Species:

Triticum aestivum

Duration:

2 wk

Dose descriptor:

LC50

Effect conc.:

> 1 000 other: mg a.i./kg soil d.w.

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: germination and survival

Effect concentrations 6

Species:

Triticum aestivum

Duration:

2 wk

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 other: mg a.i./kg soil d.w.

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: germination and survival

Details on results:

In the range finding test, there were no treatment-related effects on plant germination or survival in any of the species tested. All species and treatment levels resulted in 100% germination and 100% survival. Following statistical analysis, a significant reduction in wet weight (23%) was observed for wheat at 1000 mg a.i./Kg dry soil when compared to the solvent control. No reductions in wet weight were observed in cabbage or mung bean at any treatment level. No significant reductions in dry weight were observed for any treatment level in any species. Analysis of the control data showed no significant difference between water and solvent control groups.

In the definitive test, chemical analysis of the treatment solutions used for cabbage and mung bean, and for the highest treatment level for wheat contained 101, 102 and 101% of the nominal concentration of Citroflex® B-6, respectively. These analyses confirm correct preparation of the treatment solutions for cabbage and mung bean and the highest treatment level solution for wheat which was used to prepare the lower concentration treatment solutions.

Germination of cabbage, mung bean and wheat was 100% at all treatment levels and in all control groups.

There were no treatment-related effects on plant survival for cabbage, mung bean and wheat at any treatment level. Survival was 100% for all species and treatment levels.

There were no reductions in the wet or dry weights of cabbage, mung bean or wheat at any treatment level. There were increases in wet and dry weights in all three test species at all treatment levels tested.

Reported statistics and error estimates:

For the range finder, statistical analysis of the wet and dry weights of all species, in comparison to the solvent control, was carried out using the Williams test (Citroflex® B-6 treated groups) or t-test (water control groups versus solvent control). The wet and dry weight response was assumed to be log normally distributed. Hence the data was log transformed prior to analysis to stabilize the variance and the means were evaluated as back transformed geometric means. A significant reduction in wet weight (23%) was observed for wheat at 1000 mg a.i./Kg dry soil when compared to the solvent control. No reductions in wet weight were observed in cabbage or mung bean at any treatment level. No significant reductions in dry weight were observed for any treatment level in any species. Analysis of the control data showed no significant difference between water and solvent control groups.

For the definitive test, no statistical analysis was carried to determine the LC50 or EC50 values, as the tests with cabbage and mung bean were run as single treatment level limit tests and there was no reduction of either the wet or dry weight of wheat compared to the solvent control. Statistical analysis was carried out to determine whether the increases in wet and dry weight observed in all test species were significant using Williams test for wheat (Citroflex® B-6 treated groups) and t-tests for cabbage and mung bean Citroflex® B-6 treated groups, and for comparisons of the water controls to the solvent controls. The increases in wet and dry weights were statistically significant for both cabbage and mung bean. There were no other statistically significant differences noted for any treatment groups, or between the water and solvent control groups.

Any other information on results incl. tables

Summary of effect of Citroflex® B-6 - germination and survival:

Species	Treatment	Treatment level  (mg a.i./Kg dry soil)	Number germinated^1	Plant germination (%)	Total number of surviving plants evaluated at final assessment	Percent survival (%)
Cabbage	Water control	0	42	100	42	100
	Solvent control	0	42	100	42	100
	Citroflex^® B-6	1000	42	100	42	100
Mung bean	Water control	0	40	100	40	100
	Solvent control	0	40	100	40	100
	Citroflex^® B-6	1000	40	100	40	100
Wheat	Water control	0	42	100	42	100
	Solvent control	0	42	100	42	100
	Citroflex^® B-6	62.5	42	100	42	100
		125	42	100	42	100
		250	42	100	42	100
		500	42	100	42	100
		1000	42	100	42	100
^1 Sample size varied by plant species (n = 42 for cabbage and wheat, n = 40 for mung bean)

LC₅₀ and NOEC for each species (germination and survival):

Species	LC50  (mg a.i./Kg dry soil)	NOEC  (mg a.i./Kg dry soil)
Cabbage	>1000	≥1000
Mung bean	>1000	≥1000
Wheat	>1000	≥1000

Summary of definitive data - percent increase and reduction in wet and dry weight in comparison to the solvent control:

Species	Treatment	Treatment level  (mg a.i./Kg dry soil)	Geometric mean wet weight (mg)	% increase (+) or reduction (-) in mean wet weight ^(a)	P value	Geometric mean dry weight (mg)	% increase (+) or reduction (-) in mean dry weight ^(a)	P value
Cabbage	Water control	0	62.53	+15.4	0.057^T	3.29	+14.6	0.091^T
	Solvent control	0	54.18	-	-	2.87	-	-
	Citroflex^® B-6	1000	68.38	+26.2	0.003*^T	3.55	+23.7	0.010*^T
Mung bean	Water control	0	990.26	+8.4	0.103^T	78.68	+11.3	0.078^T
	Solvent control	0	913.29	-	-	70.72	-	-
	Citroflex^® B-6	1000	1074.29	+17.6	0.002*^T	87.42	+23.6	0.0007*^T
Wheat	Water control	0	328.95	-0.8	0.877^T	31.53	-3.4	0.530^T
	Solvent control	0	331.67	-	-	32.65	-	-
	Citroflex^® B-6	62.5	351.70	+6.0	0.272^W	34.78	+6.5	0.407^W
		125	362.63	+9.3	0.236^W	34.83	+6.7	0.407^W
		250	359.34	+8.3	0.236^W	34.52	+5.7	0.407^W
		500	346.81	+4.6	0.236^W	34.14	+4.6	0.407^W
		1000	361.28	+8.9	0.142^W	35.70	+9.3	0.146^W
Compared to the solvent control the geometric mean and % increases and reductions are taken from statistical analysis; analysis of the control data showed no significant difference between the water and solvent control groups p values are for comparison with solvent control using Williams test ^W or t-test ^T *statistically significant - not applicable

EC₅₀ and NOEC values for wet and dry weight

Species	Parameter	EC50  (mg a.i./Kg dry soil)	NOEC  (mg a.i./Kg dry soil)
Cabbage	Wet weight	>1000	>= 1000
	Dry weight	>1000	>= 1000
Mung bean	Wet weight	>1000	>= 1000
	Dry weight	>1000	>= 1000
Wheat	Wet weight	>1000	>= 1000
	Dry weight	>1000	>= 1000

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

70% emergence for control seedlings. No visible phytotoxic effects in control seedlings. 90% mean survival in controls. Environmental conditions identical for given species and same amount of soil matrix, support media or substrate from the same source.

Conclusions:

No treatment related adverse effects were observed in the seedling emergence, survival rate or growth in cabbage, mung bean or wheat, which were sown into soil treated with Citroflex® B-6 at nominal concentrations of 62.5, 125, 250, 500 and 1000 mg a.i./Kg dry soil (wheat) and 1000 mg a.i./Kg dry soil (cabbage and mung bean).
The LC50 value for the seedling emergence and survival was >1000 mg a.i./Kg dry soil in all test species. The EC50 for growth was >1000 mg a.i./Kg dry soil in all test species. The NOEC value for seedling emergence, survival and growth was determined to be ≥1000 mg a.i./Kg dry soil for all test species.
Citroflex® B-6 was therefore considered to be of low toxicity to non-target plant species.