Sodium 4(or 5)-methyl-1H-benzotriazolide

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:

2019-12-12 - 2021-06-xx

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test temperature during the study was several times below the set range of 12 -32 °C. The technical equipment of the used greenhouse did not allow a complete control of temperature. The deviations from the set range of temperature did not impact the results of this as study as shown by the performance of the control plants.

Qualifier:

according to guideline

Guideline:

OECD Guideline 208 (Terrestrial Plants, Growth Test)

Version / remarks:

2006: Terrestrial Plant Test: Seedling Emergence Test and
Commission Regulation (EU) No 2016/266 of December 07, 2015, C.31: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sponsor of Test, Batch 190733
- Purity, including information on contaminants, isomers, etc.: 99.98 % Tolyltriazole

Analytical monitoring:

no

Details on sampling:

- Sampling method: For trial 1, the application solution of the highest test item concentration was analysed. The concentration of the test item was determined by using an HPLC/UV detection system. Duplicate samples were taken and analysed.
- Sample storage conditions before analysis: frozen (about -20°C) and protected from light

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): application solution of the highest test concentration was prepared by dispersing 68447.78 mg (target 68440 mg) of the test item in 250 mL of acetone, see Table 2
- Controls: yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Evaporation of vehicle before use: Dependent on the amount of soil used for 1, 5 and 6 species, 25, 125 and 150 mL (trial 2&3: only 25mL) of the respective application solution were dropped on 50, 250 and 300 g quartz sand (trial 2&3 only 50g) and left overnight under a hood for 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: Zollinger Bio (Switzerland)
- Prior seed treatment/sterilization: untreated

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Wheat
- Plant family: Poaceae
- Source of seed: not indicated
- Prior seed treatment/sterilization: untreated

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Oilseed rape
- Plant family: Brassicaceae
- Source of seed: Saatgut Bardowick GmbH (Germany)
- Prior seed treatment/sterilization: untreated

Species:

Daucus carota

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Carrot
- Plant family: Apiaceae
- Source of seed: Zollinger Bio (Switzerland)
- Prior seed treatment/sterilization: untreated

Species:

other: Solanum lycopersicum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Tomato
- Plant family: Solanaceae
- Source of seed: Zollinger Bio (Switzerland)
- Prior seed treatment/sterilization: untreated

Species:

Phaseolus vulgaris

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Bean
- Plant family: Fabaceae
- Source of seed: Syngenta (Switzerland)
- Prior seed treatment/sterilization: untreated

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

natural soil

Remarks:

Speyer 2.1, loamy sand

Total exposure duration:

21 d

Test temperature:

9.4 – 25.4 °C (minimum-maximum) with a mean of 17.4 °C (green house cabinet 2, trial 1),
16.4 – 26.3 °C (minimum-maximum) with a mean of 20.8 °C (green house cabinet 4, trial 1),
18.2 – 28.1 °C (minimum-maximum) with a mean of 21.3 °C (green house cabinet 4, trial 2),
18.2 – 28.5 °C (minimum-maximum) with a mean of 23.2 °C (green house cabinet 4, trial 3), for one short time period below the lower limit of the recommended range of 12 – 32 °C with no impact on the outcome of the study

pH:

4.7 ± 0.1

Moisture:

31.8 ± 2.0 g per 100 g dry soil

Details on test conditions:

TEST SYSTEM
- Testing facility: Innovative Environmental Services (IES) Ltd
Benkenstrasse 260
4108 Witterswil
Switzerland
- Test container (type, material, size): non-porous plastic pots, 8x8x8 cm, surface area per pot 64cm2
- Amount of soil: 350g fresh weight
- Method of seeding: randomized design, seeds were sown per treatment and plant species after soil application, seeds were not imbibed with water. Application was performed by dissolving test item in acetone, preparing test item-sand mixtures after evaporation of the solvent and mixing the test item-sand mixtures with the soil substrates. After application, the seeds were sown.
- No. of seeds per container: 1
- No. of replicates per treatment group: 20
- No. of replicates per control: 20
- No. of replicates per vehicle control: 20

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil: Speyer 2.1
- Soil classification: loamy/silty sand
- Organic carbon (%): 0.63 ± 0.07 %
- Maximum water holding capacity (in % dry weigth): 31.8 ± 2.0 g per 100 g dry soil
- CEC: 3.7 ± 0.7 meq per 100 g
- Storage (condition, duration): in plastic bags under ambient room conditions

GROWTH CONDITIONS
- Photoperiod: 16:8
- Light source: not specified
- Light intensity and quality: 3.81-4.07 kLux (trial 1), 1.51 – 1.48 kLux (trial 2), 3.69 – 4.51 kLux (trial 3)
- Relative humidity (%): 51 - 77 % (minimum-maximum) with a mean of 64 % (green house cabinet 2, trial 1),
50 - 73 % (minimum-maximum) with a mean of 60 % (green house cabinet 4, trial 1),
47 - 78 % (minimum-maximum) with a mean of 59 % (green house cabinet 4, trial 2),
48 - 74 % (minimum-maximum) with a mean of 58 % (green house cabinet 4, trial 3), within the required limits (70 ± 25 %).
- Watering regime and schedules:
trial 1: days 0, 1, 2, 3, 8, 13, 17, 21, 24 (all) and additionally 31 (bean), 34, 36, 42 (carrot)
trial 2 (carrot): 0, 4, 7, 14, 17, 21, 25, 28
trial 3 (carrot): 0, 1, 6, 9, 14, 16, 20, 22, 26, 27
- Water source/type: local tap water for irrigation, ultarpure water for preparation of application solution
- Volume applied: 0.5 - 1.0 L tap water per tray comprising 40 pots
- Amount of soil used per vessel: 350g (fresh weight), 1 seed each

- Method of application: Prior to test start of trial 1, the application solution of the highest test concentration was prepared by dispersing 68447.78 mg (target 68440 mg) of the test item in 250 mL of acetone. The application solutions of the lower test concentrations were prepared by diluting the next higher concentration with appropriate volumes of acetone according the scheme in Table 2. Dependent on the amount of soil used for 1, 5 and 6 species, 25, 125 and 150 mL of the respective application solution were dropped on 50, 250 and 300 g quartz sand and left overnight under a hood for complete evaporation of the solvent. Afterwards, the test item-sand mixture was thoroughly mixed before the introduction into the soil substrate.
Prior to test start of trial 2 & 3, the application solution of the highest test concentration was prepared by dispersing 16689.94 (target 13688 mg) and 13687.94 mg (target 13688 mg) of the test item, respectively, in 25 mL of acetone. The application solutions of the lower test concentrations were prepared by diluting the next higher concentration with appropriate volumes of acetone according the scheme in Table 3.
25 mL of the respective application solution was dropped on 50 g quartz sand and left for four hours under a hood for complete evaporation of the solvent. Afterwards, the test item-sand mixture was thoroughly mixed before the introduction into the soil substrate.

ACCLIMATION PERIOD: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): emergence, visual phytotoxicity and mortality of emerged plants. At the end, also final fresh weight and shoot length; weekly

- Phytotoxicity rating system (if used): leaf and stem deformations, chlorosis, necrosis and overall appearance compared with control:
• 0 (no symptoms): 0% of leaf area damaged,
• 1 (slight symptoms): ≤ 25% of leaf area damaged,
• 2 (moderate symptoms): 25-75% of leaf area damaged,
• 3 (severe symptoms): ≥ 75% of area leaf damaged

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Definite test concentrations: 1.0, 3.2, 10, 32 and 100 mg/kg dry soil (onion, trial 1))
10, 32, 100, 320 and 1000 mg/kg dry soil (wheat, bean, carrot, oilseed rape and tomato, trial 1).
10, 32, 100, 320 and 1000 mg/kg dry soil (carrot, trial 2 & 3).

Nominal and measured concentrations:

nominal [mg per kg dry soil]: 10, 32, 100, 320 and 1000 mg/kg dry soil (Wheat, bean, carrot, oilseed rape, tomato in trial 1);
1.0, 3.2, 10, 32 and 100 mg/kg dry soil (Onion in trial 1);
10, 32, 100, 320 and 1000 mg/kg dry soil (Carrot in trial 2 & 3)

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

14.7 mg/kg soil dw

95% CI:

>= 6.95 - <= 22.4

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

45.7 mg/kg soil dw

95% CI:

>= 31.9 - <= 64.8

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Species:

Brassica rapa

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

7.17 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality

Species:

Brassica rapa

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

9.37 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality

Key result

Species:

Allium cepa

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.45 mg/kg soil dw

95% CI:

>= 0.28 - <= 0.65

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

shoot length

Remarks on result:

other: based on pooled control

Species:

Allium cepa

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

18.1 mg/kg soil dw

95% CI:

>= 15.7 - <= 21.3

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

shoot length

Remarks on result:

other: based on pooled control

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.91 mg/kg soil dw

95% CI:

>= 0.73 - <= 1.14

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

fresh weight

Remarks on result:

other: based on pooled control

Key result

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

9.17 mg/kg soil dw

95% CI:

>= 8.66 - <= 9.68

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

fresh weight

Remarks on result:

other: based on pooled data

Details on results:

see Table 4 and 5
- the recovery in the application solution for the highest test concentration of 1000 mg/kg dry soil was found to be 104 % of the test item

Reported statistics and error estimates:

- Fisher exact test (one-sided greater, α = 0.05) and multiple sequentially Fisher test after Bonferroni-Holm (one-sided greater, α = 0.05): emergence/mortality
- Student T-test (two-sided, α = 0.05)
- Williams t-test or the Welsh t-test for inhomogeneous variances (one-sided smaller, α = 0.05): fresh weight and shoot height per replicate
- Spearman-Karber method, moving average method and probit analysis: Calculations of EC10, EC20 and EC50 for emergence (for wheat, bean, carrot, oilseed rape and tomato) and LC10, LC20 and LC50 for mortality of emerged plants (oilseed rape only)
- Probit analysis slope/intercept: 2.94320/-6.32190 (carrot), 3.91598/-10.02242 (oilseed rape emergence), 11.03985/-10.72876 (oilseed rape mortality), 2.60264/-4.31949 (tomato)
- symptoms of toxicity were not statistically evaluated since these effects (i.e. reduced growth) were covered by the statistical analysis of growth
- non-linear regression (3-parametric normal cumulative distribution function with Downhill-Simplex method optimization and 500 optimization cycles using treatment means as values and 500 resamplings for estimating confidence limits): Calculations of EC10, EC20 and EC50 for growth (shoot length and shoot fresh weight, all species)
- In order to increase the statistical power, the calculations of wheat growth were performed excluding the treatment of 1000 mg/kg dry soil

- Statistical analysis was performed using ToxRat Professional, Version 3.3.0

Table 4: Summary Table Growth

Endpoints	Onion (A. cepa)		Wheat (T. aestivum)		Bean (P. vulgaris)	Carrot (D. carota)	Oilseed rape (B. napus)		Tomato (L. esculentum)
	Solvent control	Pooled control	Solvent control	Pooled control	Pooled control	Pooled control	Solvent control	Pooled control	Solvent control	Pooled control
Shoot Length
NOEC	<1.0	<1.0	-	<10	32	32	<10	<10	<10	<10
EC10	2.68	0.45	-	26.1	58.6	60.3	17.8	8.52	21.2	11.0
EC20	6.25	1.60	-	48.1	95.8	92.9	25.2	14.1	31.9	19.0
EC25	8.62	2.59	-	60.6	115	110	28.8	17.0	37.3	23.3
EC50	31.7	18.1	-	154	245	213	49.2	36.7	69.7	55.1
Freshweight
NOEC	10	<1.0	<10	<10	10	32	-	<10	-	<10
EC10	5.79	1.56	14.1	27.6	15.1	53.9	-	3.11	-	0.91
EC20	10.1	3.69	26.0	43.6	27.8	68.2	-	5.77	-	2.02
EC25	12.5	5.12	32.7	51.9	35.0	74.6	-	7.3	-	2.72
EC50	29.3	19.2	83.5	105	89.2	107	-	18.9	-	9.17

 

Table 5: Summary Table Emergence and Mortality

Species

Onion (A. cepa)

Wheat (T. aestivum)

Bean (P. vulgaris)

Carrot (D. carota)

Oilseed rape (B. napus)

Tomato (L. esculentum)

Endpoints

Emergence

Mortality

Emergence

Mortality

Emergence

Mortality

Emergence

Mortality

Emergence

Mortality

Emergence

Mortality

NOEC

>100

>100

320

>1000

320

>320

100

>320

100

100

10

>100

EC/LC10

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

51.6

n.a.

171

7.17

14.7

n.a.

EC/LC20

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

72.8

n.a.

221

7.86

21.7

n.a.

EC/LC25

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

82.9

n.a.

244

8.14

25.1

n.a.

EC/LC50

>100

>100

405

>1000

566

>320

141

>320

363

9.37

45.7

>100

Validity criteria fulfilled:

yes

Conclusions:

In a valid, reliable and conclusive study following the OECD 208, the effects of the test item Tolyltriazole on seedling emergence and early growth of emerged plants were tested. For the overall NOEC-values Tomato turned out to be the most susceptible species for emergence, oilseed rape for mortality, for shoot length and fresh weight it was onion (the overall EC20 value was based on tomato as the most susceptible species).

Executive summary:

The overall NOEC for emergence was based on results of tomato as the most susceptible species and was 10 mg/kg dry soil. The corresponding EC20 value was 21.7 mg/kg dry soil (95 % confidence interval: 12.1-31.1 mg/kg dry soil).

The overall NOEC for mortality of the emerged plants was based on results of oilseed rape as the most susceptible species and was 100 mg/kg dry soil. The corresponding LC20 value was 221 mg/kg dry soil (95 % confidence interval: 134-293 mg/kg dry soil).

The overall NOEC for shoot length was based on results of onion as the most susceptible species and was < 1.0 mg/kg dry soil. The corresponding EC20 value was 6.25 mg/kg dry soil (95 % confidence interval: 5.92-7.37 mg/kg dry soil) based on solvent control and 1.60 mg/kg dry soil (95 % confidence interval: 1.17-2.02 mg/kg dry soil) based on pooled control.

The overall NOEC for fresh weight was based on results of onion as the most susceptible species and was < 1.0 mg/kg dry soil. The overall EC20 value was based on tomato as the most susceptible species and was 2.02 mg/kg dry soil (95 % confidence interval: 1.73-2.37 mg/kg dry soil).

Description of key information

The toxicity to plants was investigated in a long-term study exposing five different species with Tolyltriazole. The most sensitive long-term endpoint was the shoot length in onions (EC10, 21 d: 0.45 mg/kg dw). For the growth of tomatos (shoot length) the most sensitive acute endpoint was found (EC50, 21d: 9.17 mg/kg dw).

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:

9.17 mg/kg soil dw

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

0.45 mg/kg soil dw