Benzyl-C12-14-alkyldimethylammonium chlorides

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Details on sampling:

Plants were harvested 14 - 16 days after germination by cutting each stem at soil level. The wet weight of each plant was then recorded. The plants were then dried before being re-weighed to obtain a measurement of dry weight.

Vehicle:

yes

Details on preparation and application of test substrate:

For each species, groups of 40 seeds (eight replicate pots of five seeds) were sown into a garden loam soil treated with Barquat MB 50 (ADBAC) Controls were also included. Treatment levels for the definitive study were based on the results of a preliminary range finding study.
The Alkyldimethylbenzylammonium Chloride was prepared in a 100 mL volume of reverse osmosis water for each species at each rate of application and then thoroughly mixed into the soil. A further 50 mL of water was then added to produce a soil structure suitable for sowing.
After application and sowing, the pots were checked daily and the number of seedlings emerging recorded. Survival and sub-lethal effects were recorded every day following emergence. Plants were harvested 14 - 16 days after germination and the wet weights were measured. The plants were then dried before being re-weighed to obtain a dry weight measurement.

Species:

Phaseolus aureus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Mung bean
batch: (PA0301)
Purchased ready packaged from commercial suppliers: Huntingdon Garden

Species:

Brassica alba

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Mustard seed
batch: (BA0302)
Purchased ready packaged from commercial suppliers:Frosts of Brampton respectively

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Wheat
batch: (TA 0302)
Supplied by: PBI Cambridge Ltd.

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

other: The soil used was a 50:50 w/w mix of Gem Quality Garden Top Soil (Batch GTS0303):Fibre sand (Batch FS0301)

Limit test:

no

Total exposure duration:

16 d

Test temperature:

Mung bean 22-30ºC, mustard 22-30ºC, wheat 20-24ºC

pH:

7.2

Details on test conditions:

Plants were top-watered daily until germination and then both top- and bottom-watered as required (determined on a per-species and per-treatment basis) to maintain individual seedling growth.
Environmental conditions (temperature, relative humidity and light intensity at soil level) were recorded daily throughout the exposure period.

Nominal and measured concentrations:

156, 313, 625, 1250, 2500 mg a.i./Kg dry soil of ADBAC for mung bean
12, 37, 117, 375, 1200 mg a.i./Kg dry soil of ADBAC for mustard and wheat

Reference substance (positive control):

no

Key result

Species:

Phaseolus aureus

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

1 900 mg/kg soil dw

Nominal / measured:

not specified

Conc. based on:

act. ingr.

Basis for effect:

growth

Key result

Species:

Brassica alba

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

277 mg/kg soil dw

Nominal / measured:

not specified

Conc. based on:

act. ingr.

Basis for effect:

growth

Key result

Species:

Triticum aestivum

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

670 mg/kg soil dw

Nominal / measured:

not specified

Conc. based on:

act. ingr.

Basis for effect:

growth

Details on results:

Environmental measurements
Plants were maintained in controlled environment rooms providing a continuous period of 16 hours light per day. Lighting was maintained at low levels prior to germination and then raised to facilitate growth. Temperatures were maintained at levels suitable for the individual species. A summary of environmental data is given in Table 2.

Effect on germination
Germination was recorded daily. There was no treatment-related effect on the germination of any of the plant species treated with ADBAC. For mung bean and mustard, mean germination ranged from 87.5100% in treated groups and P 95% in the controls. For wheat, germination was lower ranging from 6585% with 77.5% in the controls. Mean germination is presented in Table 3.

Effect on plant survival
The number of plants surviving at harvest and the percentage survival is given in Table 3.
There was no treatment-related effect on seedling survival for any plant species. There was 100% survival at all rates of application in mung beans. For mustard, there was 90.0 and 82.9% plant survival at 375 and 1200 mg a.i./Kg respectively and 100% survival at the lower treatment concentrations. For wheat there was 91.4% survival at 1200 mg a.i./Kg, and 100% plant survival at the lower treatment concentrations.

Effect on plant growth and health
At harvest, measurement of the wet and dry weight for each surviving seedling for each replicate pot was conducted. The geometric mean dry weights and percent reduction in comparison to the respective control are given in Table 4.
For mung bean, ADBAC at 625 mg a.i./Kg and higher gave a significant reduction (p<0.001) in dry weight, which was reduced by 29, 43 and 54% of control at 625, 1250 and 2500 mg a.i./Kg, respectively.
For mustard, ADBAC at 117 mg a.i./Kg and higher gave a significant reduction (p<0.001) in dry weight, which was reduced by 25, 64 and 74% of control at 117, 375 and 1200 mg a.i./Kg, respectively.
For wheat, ADBAC at 375 mg a.i./Kg and higher gave a significant reduction in dry weight, which was reduced by 33 and 80% of control at 375 (p<0.05) and 1200 (p<0.001) mg a.i./Kg, respectively.

A visual assessment based on the overall condition of surviving seedlings for each replicate was conducted at the end of the study and at regular intervals throughout. This assessment was conducted using a uniform scoring system based on plant size and vigor where 0 indicated no abnormal effects and 10 indicated that all plants within a replicate died. Results and descriptions of herbicidal symptoms are summarized in Table 5. These data were not included in the statistical analyses.

For all plant species growth inhibition was observed at the higher rates of application. For mung bean there was 25-40 and 50-75% inhibition at 1250 and 2500 mg a.i./Kg respectively. For mustard there was 75-80 and >80% inhibition at 375 and 1200 mg a.i./Kg respectively and 50-75% for wheat at 1200 mg a.i./Kg. Darker pigmentation was observed for all species at the higher rates of application.

Estimates and 95% confidence intervals for the point of 50% inhibition of growth (based on dry weight) are as follows:

Species	EC50 (mg a.i./Kg dry soil)	95% confidence intervals
Mung bean (Phaeolus aureus)	1900	1480, 2520
Mustard (Brassica alba)	277	222, 347
Wheat (Triticum aestivum)	670	442, 972

TABLE 1: Summary of preliminary germination trials(weight/seed and seed viability)

Species	Mean weight/seed (g)	Germination
		Trial number	Replicate number (n = 5)	Final % germination	Mean final % germination
Mung bean	0.0592 n = 10	1	1	100	96.7
			2	100
			3	100
		2	1	100
			2	100
			3	80
Mustard	0.0065 n = 20	1	1	80	88.3
			2	100
			3	100
		2	1	40
			2	100
			3	100
		3	1	100
			2	80
			3	60
		4	1	100
			2	100
			3	100
Wheat	0.0477 n = 10	1	1	100	88.3
			2	100
			3	60
		2	1	100
			2	60
			3	100
		3	1	80
			2	100
			3	80
		4	1	100
			2	80
			3	100

TABLE 2: Environmental conditions

Species	Environmental Parameters
	Air temperature (C)	Relative humidity (%)	Light (Lux)
Mung bean	22 - 30	31 - 56	274 - 4530
Mustard	22 - 30	27 - 74	223 - 5920
Wheat	20 - 24	25 - 76	100 - 3490

Lighting was maintained at low levels prior to germination and then raised to facilitate growth.

TABLE 3: Summary of effect of ADBAC germination and survival 

Species	Treatment	Soil concentration mg a.i./Kg dry soil	Mean % germination	Total number of plants evaluated at final assessment (n = 40)	% of surviving plants (as a % of germinated seedlings)
Mung bean	Water control	-	95.0	38	100
	ADBAC	156 313 625 1250 2500	92.5 92.5 97.5 100 95.0	37 37 39 39 38	100 100 100 100 100
Mustard	Water control	-	97.5	39	100
	ADBAC	12 37 117 375 1200	100 97.5 97.5 100 87.5	40 39 39 36 29	100 100 100 90.0 82.9
Wheat^b	Water control	-	77.5	30	96.8^a
	ADBAC	12 37 117 375 1200	82.5 75.0 80.0 85.0 65.0	33 28 32 33 23	100 96.5^a 100 100 91.4

^amortality due to fungal infection

^bfor wheat, correction of plant survival was calculated using Abbott s formula:   

 % survival =100 - % survival in the treatment

                     100 - % survival in the control

% survival in the control was 96.8% (30 of 31 germinated plants survived)

TABLE 4: Geometric mean dry weight and % reduction compared to the control

Species	Treatment	Soil concentration mg a.i./Kg dry soil	Mean dry weight (mg)^a	Mean % reduction compared to the control
Mung bean	Water control	-	77.6	0
	ADBAC	156 313 625 1250 2500	68.5 70.6 55.0 44.3 35.8	12 9 29*** 43*** 54***
Mustard	Water control	-	21.1	0
	ADBAC	12 37 117 375 1200	21.1 19.7 15.8 7.5 5.5	0 6 25*** 64*** 74***
Wheat	Water control	-	36.7	0
	ADBAC	12 37 117 375 1200	29.1 34.5 28.3 24.4 7.5	21 6 23 33* 80***

All statistics performed to Williams test

a- Geometric mean

* p<0.05

*** p<0.001

 

TABLE 5: Final visual assessments

Species	Treatment	Soil concentration (mg a.i./Kg dry soil)	Mean visual assessment	Description of predominant herbicidal effects in surviving plants
Mung bean	Water control	-	0.25	Very slight growth inhibition
	ADBAC	156 313 625 1250 2500	0.63 0.25 2.50 4.50 6.50	Very slight growth inhibition No effects observed Slight growth inhibition 25-40% growth inhibition^a 50-75% growth inhibition^a
Mustard	Water control	-	0.00	No effects observed
	ADBAC	12 37 117 375 1200	0.25 0.50 2.13 7.25 8.88	Very slight growth inhibition Very slight growth inhibition Slight growth inhibition 75-80% growth inhibition^a >80% growth inhibition^a
Wheat	Water control	-	0.38	1 of 31 plants dead^b
	ADBAC	12 37 117 375 1200	0.63 1.25 0.38 2.13 6.75	Very slight growth inhibition Very slight growth inhibition^b No effects observed Slight growth inhibition 50-75% growth inhibition^a

a- darker pigmentation

b- fungal infection

Scoring: 0 = no abnormal effects, to 10 = all plants within a replicate were dead.

 

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 14-16 d EC50 values based on treatment related growth inhibition in Mung beans, mustard and wheat where 1900, 277 and 670 mg a.i./Kg dry soil respectively.

Executive summary:

A study was conducted to determine the long-term toxicity of the read across substance, C12-16 ADBAC (49.5% active in water) to terrestrial plants, according to OECD Guideline 208, in compliance with GLP. Three plant species: Phaeolus aureus (mung beans) Brassica alba (mustard) and Triticum aestivum (wheat) were used. Each plant species was sown into treated soil and assessed for 14 - 16 days following germination. For each species, groups of 40 seeds (eight replicate pots of five seeds) were sown into a garden loam soil treated with the read across substance. Untreated controls were also included. Treatment levels for the definitive study were based on the results of a preliminary range finding study. The dose levels of the read across substance used were 156, 313, 625, 1250 and 2500 mg a.i./kg dry soil for mung beans and 12, 37, 117, 375 and 1200 mg a.i./kg dry soil for mustard and wheat. After application and sowing, the pots were checked daily and the number of seedlings emerging recorded. Survival and sub-lethal effects were recorded every day following emergence. Plants were harvested 14 - 16 days after germination and the wet weights were measured. The plants were then dried before being re-weighed to obtain a dry weight measurement. There was no treatment-related effect on the germination and seedling survival of any of the plant species treated with the read across substance up to the highest tested concentrations. The growth inhibition occurred at higher rates of application for all the plant species. For mung bean, there was 25-40 and 50-75% inhibition at 1250 and 2500 mg a.i./kg, respectively. For mustard, there was 75-80 and >80% inhibition at 375 and 1200 mg a.i./kg, respectively and 50-75% for wheat at 1200 mg a.i./kg. Darker pigmentation was observed for all species at the higher rates of application. Under the conditions of the study the 14-16 d EC50 values based on growth inhibition in mung beans, mustard and wheat were determined to be at 1900, 277 and 670 mg a.i./Kg dry soil respectively (Gray, 2004). Based on the results of the read across study, similar effect levels can be considered for the test substance.

Reference 2

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From November 19, 2003 to March 03, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

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)

Analytical monitoring:

yes

Remarks:

HPLC/UV

Details on sampling:

The test substance is soluble

Details on preparation and application of test substrate:

- Natural soil:
The natural soil was collected on October 10, 2003.
Sampling site 1: Mr Castagnède, Parcelle Guilhem (40110 Arengosse, France). Plant cover = peas, under agro biological practice.
Sampling site 2: Mr Dupouy, Parcelle Parille (40500 Coudures, France). Plant cover = triticales, under agro biological practice.
Both the soil samples were sieved through a 2 mm aperture size. They were mixed 50/50 w/w dry weight, and the resulting soil was sampled for further analysis. The stock soil was stored outdoors in plastic containers, which allowed free access of air.
Characteristics of the soil were as follows:
Method Results
Sieve analysis NF X31-107 Sand 48.2 %
Lime 42.8 %
Clay 9.0 %
Total water ISO 14238 holding capacity appendix A 38.3 % dry soil
pH (in water) ISO 10390 : 6.70

Catiionic exchange cap. NF X31-130 7.4 meq./100 g
Total OC NF X31-109 1.41 % dry weight
Nitrogen ISO 11261 0.084 % dw

- Sand:
Fontainebleau sand, min. 99.8% SiO2
Particle size: 150-210 µm

Species:

other: Sinapis alba; Trifolium pratense; Triticum aestivum

Plant group:

other: Dicotyledonae; Monocotyledonae

Details on test organisms:

- Cruciferae (Brassicaceae); Sinapis alba; Mustard; source: agro-biologically produced seeds ‘white mustard’, Le Biau Germe, batch 2001, expiry date : 12/2004 (47360 Montpezat d’Agenais, France)
- Leguminosae (Fabaceae); Trifolium pratense; red clover; source: agro-biologically produced seeds ‘Trèfle violet’. Le Biau Germe, batch CB1, expiry date : 12/2006 (47360 Montpezat d’Agenais, France)
- Gramineae (Poceae); Triticum aestivum; wheat; source: agro-biologically produced seeds ‘Blé tendre à germer’, Celnat, batch n° 3 273120 010304, expiry date 13/09/2004 (43700 Saint-Germain-Laprade, France)

Test type:

seedling emergence toxicity test

Study type:

laboratory study

Substrate type:

other: natural soil and quartz

Limit test:

no

Total exposure duration:

16 d

Remarks:

16d after 50% of the control plants had emerged

Test temperature:

Natural soil: Extreme values 16.0-23°C, the average daily value ranged between 17.0 and 22.0°C.
Sand: Extreme values 14.0-22.5°C, the average daily value ranged between 15.5 and 20.0°C.

Details on test conditions:

- Species: 
Monocotyledonae:  - Tritium aestivum (wheat (Category 1))
Dicotyledonae:  - Sinapis alba (mustard);  - Trifolium pratense (red clover (Category 3))
- Tested with 40 plants per replicate per dose, using 4 replicates. 
- Test was performed on natural soil and sand.
- Concentrations:   
Natural soil (mg/kg): 0, 476.6, 856.2, 1540.9, 2772.2 and 4990.0  
Sand (mg/kg): 0, 28.8, 55.8, 93.4, 166.8 and 300.5
- Endpoint/Type of test: Emergence, growth
- Design: natural soil, sand
- Duration: 16d after 50% of the control plants had emerged.

Nominal and measured concentrations:

- Natural soil (mg/kg): 0, 476.6, 856.2, 1540.9, 2772.2 and 4990.0  
- Sand (mg/kg): 0, 28.8, 55.8, 93.4, 166.8 and 300.5

Reference substance (positive control):

no

Key result

Species:

Sinapis alba

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 342 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on wet weight) in natural soil

Remarks on result:

other: Mustard; 95% confidence limits: 53-2185

Key result

Species:

Trifolium pratense

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 309 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on wet weight) in natural soil)

Remarks on result:

other: Red clover; 95% confidence limits: 151-632

Key result

Species:

Triticum aestivum

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 684 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on wet weight) in natural soil

Remarks on result:

other: Wheat; 95% confidence limits: 359-1302

Key result

Species:

Sinapis alba

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 537 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on dry weight) in natural soil

Remarks on result:

other: Mustard; 95% confidence limits: 187-1539

Key result

Species:

Trifolium pratense

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 634 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on dry weight) in natural soil

Remarks on result:

other: Red clover; 95% confidence limits: 290-1390

Key result

Species:

Triticum aestivum

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 1 960 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on dry weight) in natural soil

Remarks on result:

other: Wheat; 95% confidence limits: 758 - 5066

Key result

Species:

Sinapis alba

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

3 881 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

seedling emergence

Remarks:

natural soil

Remarks on result:

other: NOEC: 2772.2 mg/kg soil dw act. ingr. (nominal)

Key result

Species:

Triticum aestivum

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

> 4 990 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

seedling emergence

Remarks:

natural soil

Remarks on result:

other: NOEC: >4990 mg/kg soil dw act. ingr. (nominal)

Key result

Species:

Trifolium pratense

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

> 4 990 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

seedling emergence

Remarks:

natural soil

Remarks on result:

other: NOEC: 856.2 mg/kg soil dw act. ingr. (nominal)

Key result

Species:

Sinapis alba

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 31 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on wet weight) in quartz sand

Remarks on result:

other: Mustard; 95% confidence limits: 0-160

Key result

Species:

Trifolium pratense

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 19 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on wet weight) in quartz sand

Remarks on result:

other: Red clover; 95% confidence limits: 0-329

Key result

Species:

Triticum aestivum

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 105 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on wet weight) in quartz sand

Remarks on result:

other: Wheat; 95% confidence interval limits: 7-204

Key result

Species:

Sinapis alba

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 73 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on dry weight) in quartz sand

Remarks on result:

other: Mustard; 95% confidence limits: 0-99

Key result

Species:

Trifolium pratense

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 74 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on dry weight) in quartz sand

Remarks on result:

other: Red clover; 95% confidence limits: 0 - 474

Key result

Species:

Triticum aestivum

Duration:

16 d

Dose descriptor:

EC50

Effect conc.:

ca. 141 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(based on dry weight) in quartz sand

Remarks on result:

other: Wheat; 95% confidence interval limits: 78-205

Key result

Species:

Sinapis alba

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

130 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

seedling emergence

Remarks:

quartz sand

Remarks on result:

other: NOEC: 55.8 mg/kg soil dw act. ingr. (nominal)

Key result

Species:

Trifolium pratense

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

197 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

seedling emergence

Remarks:

quartz sand

Remarks on result:

other: NOEC: 93.4 mg/kg soil dw act. ingr. (nominal)

Key result

Species:

Triticum aestivum

Duration:

16 d

Dose descriptor:

LC50

Effect conc.:

234 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

seedling emergence

Remarks:

quartz sand

Remarks on result:

other: NOEC: 93.4 mg/kg soil dw act. ingr. (nominal)

Details on results:

See 'Any other information on results incl. tables' as well as the attachment under background material.

Table 1. Results (based on active test substance)

Results	wheat	mustard	red clover
Test on natural soil
LC50 (based on emergence)	> 4990.0 mg/kg	3881 mg/kg [2772.2 – 4990.0]	> 4990.0 mg/kg
EC50 (based on wet weight)	684 mg/kg [359 – 1302]	342 mg/kg [53 – 2185]	309 mg/kg [151 – 632]
EC50 (based on dry weight)	1960 mg/kg [758 – 5066]	537 mg/kg [187 – 1539]	634 mg/kg [290 – 1390]
Test on quartz sand
LC50 (based on emergence)	234 mg/kg [167 – 301]	130 mg/kg [93 – 167] mg/kg	197 mg/kg [93 – 301] mg/kg
EC50 (based on wet weight)	105 mg/kg [7 – 204]	31 mg/kg [0 – 160]	19 mg/kg [0 – 329]
EC50 (based on dry weight)	141 mg/kg [78 – 205]	73 mg/kg [0 – 99]	74 mg/kg [0 – 474]

The most sensitive was plant T. pratense with EC50 = 634 mg/kg soil dry weight.

For details on results please refer to the attachment appended under 'attached background material'.

Conclusion: The effects of test substance exhibited great deviation between sand and natural soil. In sand toxic effects take place at lower concentrations than in soil, this is probably due to the lower bioavailability of test substance in soil caused by stronger adsorption.

The test was considered as valid on the basis of percent emergence and further growth of the plant in the water control.

The side-effects exhibited great deviation between quartz sand and natural soil. The extraction of the active substance proved that the natural soil had a strong sorbing effect, and the total recovery was not achieved even when acidified methanol was used. That was not the case with quartz sand, and this was taken as the source of the differences: the availability of the active substance for the plants differed, depending on the nature of the soil substrate.

Validity criteria fulfilled:

yes

Remarks:

The test was considered as valid on the basis of percent emergence and further growth of the plant in the water control.

Conclusions:

Under the conditions of the study, based on effect on emergence and growth (based on wet weight red clover was identified to be the most sensitive species with lower NOEC and EC50 value of 856.2 and 309 mg a.i./kg dw soil respectively.

Executive summary:

A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49.9% active in water) to terrestrial plants, according to OECD Guideline 208, in compliance with GLP. Three plant species:Sinapis alba(mustard),Trifolium pratense(red clover) andTriticum aestivum(wheat) were used. Using 0.5 L capacity plastic pots, the read across substance was first applied to natural soil at nominal concentrations of 0, 476.6, 856.2, 1540.9, 2772.2 and 4990.0 mg a.i./kg and to sand at nominal concentrations of 0, 28.8, 55.8, 93.4, 166.8 and 300.5 mg a.i./kg. This was followed by planting of 40 seeds per replicate of the three plant species. Analytical verification was performed for the read across substance. Three parameters: emergence, dry and wet weight of the plants were observed. Emergence was recorded daily until stabilisation. The plants in natural soil and sand were harvested 16 and 14 d respectively after 50% of the control seeds had been emerged. Wet and dry weight were determined immediately after harvesting. The test was considered as valid on the basis of percent emergence and further growth of the plant in the water control. The extraction of the active substance proved that the natural soil had a strong sorbing effect and the total recovery was not achieved even when acidified methanol was used as an extraction solvent. That was not the case with quartz sand. The LC50 values in natural soil based on effect on emergence were 3881, >4990 and >4990 mg a.i./kg dw of soil for mustard, red clover and wheat respectively; while those in sand were 130, 197, 234 mg a.i./kg dw soil. The corresponding NOEC values were 2772.2, 856.2, >4990 mg a.i./kg dw in natural soil and 55.8, 93.4 and 93.4 mg a.i./kg dw in sand. The EC50 values in natural soil, based on the effect on growth were 342, 309, 684 mg a.i./kg dw of soil (based on changes in wet weight) and 537, 634 and 1960 mg a.i./kg dw of soil (based on changes in dry weight) for mustard, red clover and wheat respectively, respectively; while those in sand were 31, 19, 105 mg a.i./kg dw of soil (based on changes in wet weight) and 73, 74 and 141 mg a.i./kg dw soil (based on changes in dry weight) of sand respectively. The difference in toxicity in the two substrates were correlated with the lower bioavailability of read across substance in soil due to a stronger adsorption potential. Further, as the toxicity to terrestrial plants in sand is not representative of the natural environment, the EC50 in natural soil was considered as a reasonable worst case for representing toxicity terrestrial plant species. Under the conditions of the study, based on effect on emergence and wet weight changes (growth) red clover was identified to be the most sensitive species with lower NOEC and EC50 value of 856.2 and 309 mg a.i./kg dw soil respectively (Servajean, 2004). Based on the results of the read across study, similar effect levels can be considered for the test substance.

Reference 3

Endpoint:

toxicity to terrestrial plants: long-term

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Description of key information

Based on the results of the read across study and in line with the C12-16 ADBAC biocides assessment report, the 16-d EC50 value of 277 mg a.i./kg dw of soil obtained forBrassica alba(mustard) due to effects on growth has been considered further for hazard/risk assessment. Additionally, in accordance with the ECHA R.7c guidance (2017), the 16-d NOEC value of 856.2 mg a.i./kg dw of soil obtained forTrifolium pratense(red clover) due to effects on growth can be considered as the long-term equivalent value.

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:

277 mg/kg soil dw

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

856.2 mg/kg soil dw

Additional information

Study 1:A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49.9% active in water) to terrestrial plants, according to OECD Guideline 208, in compliance with GLP. Three plant species:Sinapis alba(mustard),Trifolium pratense(red clover) andTriticum aestivum(wheat) were used. Using 0.5 L capacity plastic pots, the read across substance was first applied to natural soil at nominal concentrations of 0, 476.6, 856.2, 1540.9, 2772.2 and 4990.0 mg a.i./kg and to sand at nominal concentrations of 0, 28.8, 55.8, 93.4, 166.8 and 300.5 mg a.i./kg. This was followed by planting of 40 seeds per replicate of the three plant species. Analytical verification was performed for the read across substance. Three parameters: emergence, dry and wet weight of the plants were observed. Emergence was recorded daily until stabilisation. The plants in natural soil and sand were harvested 16 and 14 d respectively after 50% of the control seeds had been emerged. Wet and dry weight were determined immediately after harvesting. The test was considered as valid on the basis of percent emergence and further growth of the plant in the water control. The extraction of the active substance proved that the natural soil had a strong sorbing effect and the total recovery was not achieved even when acidified methanol was used as an extraction solvent. That was not the case with quartz sand. The LC50 values in natural soil based on effect on emergence were 3881, >4990 and >4990 mg a.i./kg dw of soil for mustard, red clover and wheat respectively; while those in sand were 130, 197, 234 mg a.i./kg dw soil. The corresponding NOEC values were 2772.2, 856.2, >4990 mg a.i./kg dw in natural soil and 55.8, 93.4 and 93.4 mg a.i./kg dw in sand. The EC50 values in natural soil, based on the effect on growth were 342, 309, 684 mg a.i./kg dw of soil (based on changes in wet weight) and 537, 634 and 1960 mg a.i./kg dw of soil (based on changes in dry weight) for mustard, red clover and wheat respectively, respectively; while those in sand were 31, 19, 105 mg a.i./kg dw of soil (based on changes in wet weight) and 73, 74 and 141 mg a.i./kg dw soil (based on changes in dry weight) of sand respectively. The difference in toxicity in the two substrates were correlated with the lower bioavailability of test substance in soil due to a stronger adsorption potential. Further, as the toxicity to terrestrial plants in sand is not representative of the natural environment, the EC50 in natural soil was considered as a reasonable worst case for representing toxicity terrestrial plant species. Under the conditions of the study, based on effect on emergence and wet weight changes (growth) red clover was identified to be the most sensitive species with lower NOEC and EC50 value of 856.2 and 309 mg a.i./kg dw soil respectively (Servajean, 2004).    

Study 2:  A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49.5% active in water) to terrestrial plants, according to OECD Guideline 208, in compliance with GLP. Three plant species:Phaeolus aureus(mung beans)Brassica alba(mustard) andTriticum aestivum(wheat) were used. Each plant species was sown into treated soil and assessed for 14 - 16 days following germination. For each species, groups of 40 seeds (eight replicate pots of five seeds) were sown into a garden loam soil treated with the read across substance. Untreated controls were also included. Treatment levels for the definitive study were based on the results of a preliminary range finding study. The dose levels of the read across substance used were 156, 313, 625, 1250 and 2500 mg a.i./kg dry soil for mung beans and 12, 37, 117, 375 and 1200 mg a.i./kg dry soil for mustard and wheat. After application and sowing, the pots were checked daily and the number of seedlings emerging recorded. Survival and sub-lethal effects were recorded every day following emergence. Plants were harvested 14-16 days after germination and the wet weights were measured. The plants were then dried before being re-weighed to obtain a dry weight measurement. There was no treatment-related effect on the germination and seedling survival of any of the plant species treated with the read across substance up to the highest tested concentrations. The growth inhibition occurred at higher rates of application for all the plant species. For mung bean, there was 25-40 and 50-75% inhibition at 1250 and 2500 mg a.i./kg, respectively. For mustard, there was 75-80 and >80% inhibition at 375 and 1200 mg a.i./kg, respectively and 50-75% for wheat at 1200 mg a.i./kg. Darker pigmentation was observed for all species at the higher rates of application. The 14-16 d EC50 values based on growth inhibition in mung beans, mustard and wheat were determined to be1900, 277 and 670 mg a.i./Kg dry soil respectively. Under the conditions of the study, based on effect on growth, mustard was identified to be the most sensitive species with lower EC50 value of 277 mg a.i./kg dw soil (Gray, 2004). 

Based on the above studies, same effect levels and low toxicity potential were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. They further stated that:“The great deviation in the effects recorded in sand and natural soil can be attributed to the lower bioavailability of C12-16 ADBAC in natural soil caused by stronger adsorption to the soil particles as consequence of several binding processes. Since the results obtained in the test with silica sand are considered unrealistic worst case, only data from the tests conducted with natural soils are taken into account (this approach was agreed at TMII2013); among these, the most sensitive species was Brassica alba with an EC50 = 277 mg/kg dw soil (US ISC), which is the endpoint to be taken into account at product authorization stage” (ECHA biocides assessment report, 2015).   

 In line with the C12-16 ADBAC biocides assessment report and given that the read across to C12-16 ADBAC can be justified for the test substance based on a category approach, the 16-d EC50 value of 277 mg a.i./kg dw of soil obtained for Brassica alba (mustard) due to effects on growth has been considered further for hazard/risk assessment. Additionally, in accordance with the ECHA R.7c guidance (2017), the 16-d NOEC value of 856.2 mg a.i./kg soil dw obtained for Trifolium pratense(red clover) due to effects on growth can be considered as the long-term equivalent value.