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Toxicity to terrestrial plants

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Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Dec 2014 - 18 Jun 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
ISO 22030 (Soil quality - Biological methods - Chronic toxicity in higher plants)
Deviations:
yes
Remarks:
The volume of test solutions was not sufficient to take analytical samples of at least 50 mL per sample. Since much smaller samples were still sufficient for chemical analysis, this deviation has no impact on the integrity of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
no
Remarks:
Concentration was only measured in the stock solution before application.
Details on sampling:
Sample preparation: dilution with acetonitrile
Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): A stock solution and respective test solutions for each treatment were prepared freshly on the day of application. 32 g of the test item were diluted with acetone to obtain test solutions of 100 mL, each. A volume of 20 mL of the respective test solution was the applied to 64 g of quartz sand. After 3 h of evaporation at air temperature of 19.8 - 21.5 °C the spiked sand was mixed thoroughly into 6.4 kg of test soil.
- Controls: yes, natural soil without test substance application
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Evaporation of vehicle before use: yes
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: oat
- Plant family: Poaceae
- Variety: "Champion"
- Source of seed: Saaten-Union GmbH, Isemhagen, Germany
Species:
Brassica rapa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: turnip rape
- Plant family: Brassicaceae
- Variety: "Wisconsin fast plant"
- Source of seed: Carolina Biological Supply Company, Burlington NC, USA.
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
46 d
Remarks:
Duration of the test: 35 d for Brassica rapa and 46 d for Avena sativa
Test temperature:
15.5 - 25.7 °C (mean: 22.2 °C)
pH:
soil pH: 5.8
Moisture:
5.7% of dry weight at test start
Details on test conditions:

Observation periods and duration of test
Test period: 46 d for Avena sativa, 35 d for Brassica rapa. The emergence was assessed 7 days after seedling. Endpoints were determined after 14 d and 35 and 46 d, respectively.

TEST SYSTEM
- Testing facility: ECT Oekotoxikologie GmbH, Flörsheim, Germany
- Test container (type, material, size): Common plastic plant pots (12x12x12 cm). Each pot was placed in a rectangular polystyrene beaker (18.3x13.6x6.1 cm).
- Amount of soil: 850 ± 10 g soil ww
- Method of seeding: Test species were sown per pot using tweezers.
- No. of seeds per container: 12
- No. of plants (retained after thinning): Thinning to 8 seedlings per pot on day 7.
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Natural field soil, Lufa standard soil type 2.3 (batch Sp2.33914)
- Pesticide use history at the collection site: none since 2010
- Sampling depth (cm): 20 cm
- Soil texture (if natural soil)
- % sand: 59.9 ± 1.9
- % silt: 33.8 ± 0.2
- % clay: 6.3 ± 1.9
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- Organic carbon (%): 0.67 ± 0.04
- Maximum water holding capacity (in % dry weigth): 35.6 ± 1.7 g/100 g
- CEC: 7.3 ± 1.0 meq/100 g
- Pretreatment of soil: Air drying (only until sieveable) from Sep 24 to Sep 30 2014, final sieving to 2 mm on Oct 01, 2014.

GROWTH CONDITIONS
- Photoperiod: 16 hours light : 8 hours dark
- Light source: artificial light
- Light intensity and quality: 206 - 275 µE m-2 s-1
- Day/night temperatures: 16 °C to 26 °C
- Relative humidity (%): Mean relative air humidity was 34% (range: 22% to 55%)
- Watering regime and schedules: Water and nutrient medium was added to the plants as needed.
- Volume applied: ad libitum
- Method of application: bottom watering

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
On day 14 of the test, four out of the eight plants per pot were harvested (the plants were cut off at the soil surface) and the following endpoints were measured:
- occurrence of visible flower buds per plant (only Brassica rapa) (i.e. yes/no determination);
- number of flowers per plant (only Brassica rapa);
- shoot fresh weight per plant;
- proportion of living plants (percentage of plants compared to the number after thinning out);
- number of damaged (chlorotic, crippled, etc.) plants (qualitatively).

From day 14 to day 35, flowers of B. rapa were pollinated by hand on day 14, 16, 18, 21, 23, 24, 28, and 30.
On day 35 (Brassica rapa) and on day 46 (Avena sativa), the remaining four plants per pot were harvested. This was done by clipping each single plant at the soil surface with a scissors.
The following endpoints were determined:
- growth stage according to the BBCH scale (BBA 2001); number of all inflorescences per plant (A. sativa only);
- number of seed pods carrying fertile seeds (visibly swollen) per plant (B. rapa only);
- fresh weight of shoots (A. sativa: without inflorescences; B. rapa: without seed pods) per plant;
- fresh weight of inflorescences (A. sativa) or seed pods (B. rapa) per plant;
- dry weight of shoots (A. sativa: without inflorescences; B. rapa: without seed pods); dry weight of - inflorescences (A. sativa) or seed pods (B. rapa);
- proportion of dead plants (percentage of plants compared to the number after thinning out).

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: control, solvent control, 0.1, 1.0, 10, 100, 1000 mg/kg soil dw
- Results used to determine the conditions for the definitive study: At test item concentrations up to and including 100 mg/kg soil dry weight no effects on either shoot length or fresh weight of any of the two test species A. sativa and B. rapa were observed. At the highest tested concentration of 1000 mg/kg soil dw no seedling emerged.
Nominal and measured concentrations:
Nominal dosages: control, solvent control, 12.5, 25.0, 50.0, 100, 200, 400 mg/kg soil dw

Nominal concentrations of stock solution: 128, 64, 32, 16, 8 mg/mL
Measured concentrations of stock solution: 130, 66.4, 33.2, 14.3, 8.20 mg/mL
The stock solution of the lowest test concentration was not analysed since it was accidentally lost.
Reference substance (positive control):
no
Species:
Avena sativa
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Avena sativa
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Brassica rapa
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Brassica rapa
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Species:
Brassica rapa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight and number of flowers
Species:
Avena sativa
Duration:
46 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh and dry weight of inflorescences
Species:
Avena sativa
Duration:
46 d
Dose descriptor:
NOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh and dry weight, number of inflorescences
Species:
Avena sativa
Duration:
46 d
Dose descriptor:
LOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh and dry weight of inflorescences
Species:
Brassica rapa
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot dry weight and fresh and dry weight of inflorescences
Species:
Brassica rapa
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight and number of inflorescences
Species:
Brassica rapa
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot dry weight, fresh and dry weight of inflorescences
Details on results:
Avena sativa
- Seedling emergence: No effects on seedling emergence were recorded after 7 d of exposure compared to the control and solvent control up to 200 mg/kg soil dw. At 400 mg/kg soil dw no emergence was recorded.
- Shoot fresh weight: After 14 d the shoot fresh weight was significantly smaller compared to the solvent control at 200 mg/kg soil dw. After 46 d no significant change in shoot fresh weight was recorded up to 200 mg/kg soil dw. At 400 mg/kg soil dw no plant emerged.
- Shoot dry weight: There was no significant difference between the solvent control and any concentration tested up to 200 mg/kg soil dw after 46 d. At 400 mg/kg soil dw no plant emerged.
- BBCH growth stage: On day 46 all plants were in growth stage 58-61 of the BBCH scale except for 200 mg/kg soil dw (BBCH: 49-57). At 400 mg/kg soil dw no plant emerged.
- Number of inflorescences: There was no significant effect on the number of inflorescences up to 200 mg/kg soil dw. However, the mean number of inflorescences at 200 mg/kg soil dw was clearly below the mean of the other treatments (13.4 compared to 34.2 - 37.3). Due to the high variance a significant change could not be detected. At 400 mg/kg soil dw no plant emerged.
- Fresh weight of inflorescences: The mean fresh weight of inflorescences was significantly reduced at 200 mg/kg soil dw compared to the solvent control on day 46 (0.54 g compared to 1.80 g).
Dry weight of inflorescences: The mean dry weight of inflorescences was significantly reduced at 200 mg/kg soil dw compared to the solvent control on day 46 (0.62 g compared to 2.23 g).

Brassica rapa
- Seedling emergence: No seedling emerged at the two highest concentrations of 200 and 400 mg/kg soil dw after 7 d.
- Shoot fresh weight: The shoot fresh weight after 14 d was not significantly different to the solvent control up to 100 mg/kg soil dw. At 200 and 400 mg/kg soil dw no plants emerged after 14 d. On day 35 the shoot fresh weight did not differ significantly from the control up to 200 mg/kg soil dw.
- Shoot dry weight: On day 35 the shoot dry weight was significantly smaller compared to the solvent control at 200 mg/kg soil dw. At 400 mg/kg soil dw no plants emerged.
- Number of flowers: On day 14 all plants up to and including 100 mg/kg soil dw showed flowers and/or flower buds.
- BBCH growth stage: On day 35 all plants were in growth stage 73-79 of the BBCH scale except for 200 mg/kg soil dw (BBCH: 65-75). At 400 mg/kg soil dw no plant emerged.
- Number of seed pods: There was no significant effect on the number of seed pods up to 200 mg/kg soil dw. However, the mean number of seed pods at 200 mg/kg soil dw was clearly below the mean of the other treatments (9.33 compared to 14.92 - 17.55). Due to the high variance a significant change could not be detected. At 400 mg/kg soil dw no plant emerged.
- Seed pod fresh weight: The fresh weight of seed pods at 200 mg/kg soil dw was significantly smaller compared to the solvent control after 35 d (0.87 g compared to 2.01 g).
- Seed pod dry weight: The dry weight of seed pods at 200 mg/kg soil dw was significantly smaller compared to the solvent control after 35 d (0.11 g compared to 1.68 g).
Reported statistics and error estimates:
Statistical evaluation was either performed on the basis of the arithmetic means per plant (shoot fresh weight, number of flowers, number of fresh weight of inflorescences, number and fresh weight of seed pods), or on the basis of the sum per pot (number of seedlings, shoot dry weight, dry weight of A. sativa inflorescences and dry weight of B. rapa seed pods). Normal distribution was tested by Kolmogorov-Smirnov test, homogeneity of variances by the Cochran’s test. NOECs were either determined by applying Student’s t-test procedure with Bonferroni-Holm adjustment (p<0.05, one-sided smaller) or, if homogeneity of variance was denied, the Welch t-test for inhomogeneous variances with Bonferroni adjustment (p<0.05, one-sided smaller). Statistical evaluations were performed using ToxRat v2.1.

Table 1: NOEC/LOEC values derived for Avena sativa

Endpoint

NOEC [mg/kg soil dw]

LOEC [mg/kg soil dw]

Seedling emergence until day 7

200

400

Shoot fresh weight on day 14

100

200

Shoot fresh weight on day 46

200

n.a.

Shoot dry weight on day 46

200

n.a.

Number of inflorescences on day 46

200

n.a.

Fresh weight of inflorescences on day 46

100

200

Dry weight of inflorescences on day 46

100

200

n.a.: not available (no seedlings at 400 mg/kg soil dw)

Table 2: NOEC/LOEC values derived for Brassica rapa

Endpoint

NOEC [mg/kg soil dw]

LOEC [mg/kg soil dw]

Seedling emergence until day 7

100

200

Shoot fresh weight on day 14

100

n.a.

Number of flowers on day 14

100

n.a.

Shoot fresh weight on day 35

200

n.a.

Shoot dry weight on day 35

100

200

Number of seed pods on day 35

200

n.a.

Seed pod fresh weight on day 35

100

200

Seed pod dry weight on day 35

100

200

 

The validity criteria for the controls as defined by the test guideline have been fulfilled:

- The emergence rate in the untreated control soil was 95.8% (A. sativa) and 97.9% (B. rapa). At least 75% are required according to the guideline.

- Healthy plants developed. Plants did not etiolate and flowers appeared during the first eight weeks (A. sativa) or three weeks (8. rapa).

- Not more than one of the emerged plants died during the test.

Validity criteria fulfilled:
yes
Remarks:
see "Any other information on results incl. tables"
Conclusions:
Overall NOEC: 100 mg/kg soil dw (Brassica rapa and Avena sativa)
Executive summary:

The available study was conducted according to ISO 22030 (GLP). Seeds of Brassica rapa were exposed for 35 d whereas seeds of Avena sativa were exposed for 46 d to different concentrations of the test item. The study was conducted in a growth chamber under controlled test conditions. The test substance completely inhibited the emergence of A. sativa and B. rapa at a concentration of 400 mg/kg soil dw. At 200 mg/kg soil dw the emergence of B. rapa was still strongly retarded. Reductions in shoot weight and in the weight of inflorescences and seed pods were seen at concentration of 200 mg/kg soil dw. No adverse effects were observed at test concentrations up to 100 mg/kg soil dw. The overall lowest NOEC was 100 mg/kg soil dw.

Endpoint:
toxicity to terrestrial plants: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Nov 2006 - 11 Dec 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The source and history of the seed, the description of seed storage, treatment and maintenance are missing. The analytical verification is not clearly presented.
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants, Growth Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The homogeneity of the active ingredient in the highest concentration was verified analytically.
- Sampling method: samples were taken in duplicate
- Sample storage conditions before analysis: The samples were stored deep frozen ( < -10 °C) until analysis was performed.
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): Adequate weights of test item were diluted in 8 to 22 mL acetone. Then the corresponding dilutions were added to 638.6 to 1756.2 g fine quartz sand (0.0125 mL acetone/1 g sand). After mixing with a laboratory mixer (10 minutes) to reach a homogeneous distribution of the test item within the sand the treated sand was left overnight in a fume hood until the solvent had evaporated.
- Controls: blank and vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Evaporation of vehicle before use: yes
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: rape
- Plant family: Brassicaceae
Species:
Glycine max (G. soja)
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: soy bean
- Plant family: Fabaceae
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: oat
- Plant family: Poaceae
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Test temperature:
24.6 °C (range 20.3-29 °C)
Moisture:
56.5 % (range 35.7-80.7 %)
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): commercial plastic pots (diameter: 12 cm and 14 cm)
- No. of seeds per container: 5 seeds
- No. of replicates per treatment group: 6
- No. of replicates per control: 6
- No. of replicates per vehicle control: 6

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Source: The soil was delivered and analysed by LUFA Speyer, Germany.
- Type: LUFA 2.3 (USDA: sandy loam)
- Organic carbon (%): 1.02 ± 0.15%

GROWTH CONDITIONS
- Photoperiod: 16 h light: 8 h dark
- Light intensity and quality: 14562 lux (5690-20140 lux)
- Relative humidity (%): 56.5 % (range 35.7-80.7 %)
- Method of application: The seeds were introduced manually into the treated soil.
- Any pest control method/fertilization (if used): After development of the first true leaves, 1 g/L Flory 9 “Hydro” (Planta) and 0.022 g/L Fetrilon 13 % (Compo) were added to the water one to three times a week, depending on the development of the plants.

ACCLIMATION PERIOD: 22 Nov - 23 Nov 2006

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Fresh Weight: The fresh weight of all survived plants of a pot ( each pot is considered as a replicate) was determined 14 days after 50 % seedling emergence in the control.
Germination: Germination was checked daily until 50% of the control plants had emerged. Further checks were done weekly.
Mortality: The number of living and dead plants was recorded 14 days after 50 % seedling emergence in the control. A plant was considered dead if no living tissue could be found on the leaves or shoots. All other plants were considered living.
Phytotoxicity: Visual phytotoxicity ( e.g. chlorosis, necrosis, abnormal growth) was recorded weekly
Growth Stages: Growth stages at day 14 after 50 % seedling emergence in the control was reported according to BBCH-Monograph - Growth stages.

- Phytotoxicity rating system (if used):
Visual phytotoxicity ( e.g. chlorosis, necrosis, abnormal growth) was recorded weekly according to EPPO Standard PP 1/135 (2) after 50% seedling emergence in the control.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.96 mg test item/kg, 2.40 mg test item/kg, 6.00 mg test item/kg, 15.0 mg test item/kg, 37 .5 mg test item/kg, 93.8 mg test item/kg, 234.4 mg test item/kg
Nominal and measured concentrations:
Brassica napus:
nominal: control, 0.96, 2.40, 6.00, 15.0, 37.5, 93.8, 234.4 mg test item/kg dw
Glycine max:
nominal: control, 2.40, 6.00, 15.0, 37.5, 93.8, 234.4 mg test item/kg dw
Avena sativa:
nominal: control, 15.0, 37.5, 93.8, 234.4 mg test item/kg dw
Reference substance (positive control):
no
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
54.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks on result:
other: The result was normalized and converted to a standard soil (3.4% organic carbon + Fom 1.734%)
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
27.7 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks on result:
other:
Remarks:
95 % CI: 25.3 - 27.7 mg/kg soil dw
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
15 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Species:
Glycine max (G. soja)
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
62.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks on result:
other:
Remarks:
95 % CI: 2.65 - 30536 mg/kg soil dw
Species:
Glycine max (G. soja)
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
30.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks on result:
other:
Remarks:
95 % CI: 27.7 - 33.2 mg/kg soil dw
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
< 15 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Details on results:
Brassica napus
SEED GERMINATION
- Percent seed germination: The germination rate ranged between 0% and 100% (solvent control: 97%). A statistically significant reduction was observed at 93.8 mg/kg and 234.4 mg/kg (73% and 0 %).

FRESH WEIGHT
- The fresh weight was statistically significantly reduced at 37.5 mg/kg and 93.8 mg/kg (70.0% and 99.6 %).

The plant development was delayed at 37.5 mg/kg and 93.8 mg/kg (growth stages at test end: BBCH 14 in the controls, BBCH 12 to 13 at 37.5 mg/kg and BBCH 09 to 10 at 93.8 mg/kg).

MORTALITY
- Mortality was observed at 93.8 mg/kg (45 %).

PHYTOTOXICITY
- Phytotoxic effect was growth reduction at 37.5 mg/kg and 93.8 mg/kg. Additionally chlorosis and necrosis were observed at 93.8 mg/kg. There was a slight chlorosis in one pot each at 2.40 mg/kg and 0.96 mg/kg.

Glycine max
SEED GERMINATION
- Percent seed germination: The germination rate ranged between 10% and 100% (solvent control: 100%). A statistically significant reduction was observed at 234.4 mg/kg (10 %).

FRESH WEIGHT
- The fresh weight was reduced at all tested rates (7.0% to 99.5%). This reduction was statistically significant at 37.5 mg/kg and at higher rates (29.8% to 99.5%). The effect at 6.00 mg/kg is considered to be not test item related as at 15.0 mg/kg the fresh weight reduction was statistically not significant.

The plant development was delayed from 93.8 mg/kg onwards (growth stages at test end: BBCH 13 to 14 in the controls, BBCH 13 at 93.8 mg/kg and BBCH 09 at 234.4 mg/kg).

MORTALITY
- No statistically significant mortality occurred.

PHYTOTOXICITY
- Phytotoxic effect were chlorosis and growth reduction at 93.8 mg/kg. At 234.4 mg/kg a strong growth reduction was observed. Because one seedling was dead in one pot at 2.40 mg/kg there was an overall phytotoxic effect of 3.3 % for this concentration.

Avena sativa
SEED GERMINATION
- Percent seed germination: The germination rate ranged between 10% and 97% (solvent control: 93%). A statistically significant reduction was observed at 234.4 mg/kg (10%).

FRESH WEIGHT
- The fresh weight was statistically significantly reduced at all tested concentrations (15.1 % to 99.9%).

The plant development was delayed compared to the control at 37.5 mg/kg and at higher concentrations (growth stages at test end: BBCH 13 in the controls, BBCH 12 at 37.5 mg/kg, BBCH 10 to 11 at 93.8 mg/kg and BBCH 10 at 234.4 mg/kg).

MORTALITY
- Mortality was observed at 234.4 mg/kg (33 %).

PHYTOTOXICITY
- Phytotoxic effects were chlorosis, necrosis and growth reduction.
Reported statistics and error estimates:
Statistical analysis of the control plants and solvent control plants:
Fresh weight data of the control and solvent control plants were tested for normal distribution and homogeneity of variance using the Kolmogoroff-Smirnov-Test and the Cochran-Test. Because the data were normally distributed and homogeneous the Student-t-test was used. For the mortality and germination data Fisher's Exact-Test was used.Because there was no statistical difference between the data ofthe control plants and solvent control plants the results of the plants treated with the tested concentrations were compared with the results of the solvent control plants. Statistical analysis of the plants treated with the tested concentrations and the solvent control plants: Fresh weight data were tested for normal distribution and homogeneity of variance using the Kolmogoroff-Smirnov-Test and the Cochran-Test. If the data were normally distributed and homogeneous the Dunnett Test was used for comparing treatment groups and control. If the data were not homogeneous the Bonferroni-Welch-t-test was used. In order to determine the EC25 and EC50 values, a regression analysis was performed (Probit-analysis). For the mortality and germination data Fisher's Exact-Test was used. The computer programs used to perform the statistical analyses were ToxRat® SPiRiT Solutions (2005), Version 2.09 The significance level for all tests was a= 0.05 (two-sided).

Table 1: Effective phytotoxicity after test termination (Part 1)

 

Germination

Mortality

Fresh weight

 

Treatment Group
(mg test item/kg dry soil) (nominal)

(%)

Statistics

(%)

Statistics

(g)

SD

Effect (%)*

Statistics

Time of Evaluation**

Species

Day 14

 

Day 14

 

Day 14

 

 

 

Brassica napus

Control

90

 

0

 

10.44

1.55

 

 

Solvent Control

97

 

0

 

10.22

1.28

 

 

0.96

100

n.s.3

0

n.s.3

9.90

0.93

-3.1

n.s.1

2.40

97

n.s.3

3

n.s.3

10.20

1.65

-0.2

n.s.1

6.00

97

n.s.3

0

n.s.3

10.24

1.08

0.1

n.s.1

15.0

97

n.s.3

0

n.s.3

8.75

1.17

-14.4

n.s.1

37.5

93

n.s.3

0

n.s.3

3.07

0.62

-70.0

s.1

93.8

73

s.3

45

s.3

0.04

0.03

-99.6

s.1

234.4

0

s.3

-

-

-

-

-

-

Glycine max

Control

100

 

0

 

17.89

± 1.59

 

 

Solvent Control

100

 

0

 

17.19

± 1.05

 

 

2.40

100

n.s.3

3

n.s.3

15.99

± 2.52

-7.0

n.s.2

6.00

93

n.s.3

0

n.s.3

12.88

± 3.23

-25.1

s.2

15.0

100

n.s.3

0

n.s.3

14.71

± 1.90

-14.4

n.s.2

37.5

100

n.s.3

0

n.s.3

12.07

± 1.30

-29.8

s.2

93.8

93

n.s.3

0

n.s.3

8.31

± 0.85

-51.6

s.2

234.4

10

s.3

0

n.s.3

0.08

± 0.03

-99.5

s.2

Avena sativa

Control

97

 

0

 

9.30

± 0.45

 

 

Solvent Control

93

 

0

 

8.57

± 0.90

 

 

15.0

87

n.s.3

0

n.s.3

7.28

± 2.02

-15.1

s.2

37.5

93

n.s.3

0

n.s.3

3.28

± 0.89

-61.7

s.2

93.8

97

n.s.3

0

n.s.3

0.24

± 0.06

-97.2

s.2

234.4

10

s.3

33

n.s.3

0.01

± 0.00

-99.9

s.2

The results represent rounded values calculated on the exact raw data

SD    Standard Deviation

*       negative values indicate reduction compared to the solvent control

**     days after 50 % seedling emergence in the control

-       not evaluated, because there was no germination

1       Multiple comparison Dunnett Test, α = 0.05, two sided

2       Multiple comparison Bonferroni Welch-t-Test, α = 0.05, two sided

3       Pairwise comparison Fisher Exact Test, α = 0.05, two sided

s.       significant

n.s.    not significant

Table 2: Effective phytotoxicity after test termination (Part 2)

 

Phytotoxicity (%)

Growth Stage (BBCH)

Time of Evaluation*

Species

Treatment Group
(mg test item/kg dry soil) (nominal)

Day 7

Day 14

Day 14

Brassica napus

Control

0

0

14

Solvent Control

0

0

14

0.96

0

1

14

2.40

1

2

13-14

6.00

0

0

14

15.0

0

0

14

37.5

20

23

12-13

93.8

52

95

09-10

234.4

-

-

-

Glycine max

Control

0

0

13-14

Solvent Control

0

0

13-14

2.40

0

3

13-14

6.00

0

0

13-14

15.0

0

0

13-14

37.5

3

0

13-14

93.8

28

11

13

234.4

60

97

09

Avena sativa

Control

0

0

13

Solvent Control

0

0

13

15.0

2

2

13

37.5

38

16

12

93.8

63

83

10-11

234.4

89

98

10

**days after 50 % seedling emergence in the control

Table 3: Validity criteria

Criterion from the guideline to be satisfied in the controls

Outcome

Validity criterion fulfilled

The seedling emergence is at least 70%.

90 - 100 %

yes

The seedlings do not exhibit visible phytotoxic effects (e.g. chlorosis, necrosis, wilting, leaf and stem deformations) and the plants exhibit only normal variation in growth and morphology for that particular species.

Growth and morphology of the control seedlings: the control seedlings exhibited only normal variation in growth and morphology for that particular species

yes

The mean survival of emerged control seedlings is at least 90% for the duration of the study.

100 %

yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
Brassica napus: (14 d) EC50 = 27.7 mg/kg dw; 54.31 mg/kg dw (normalised to standard soil)
Glycine max: (14 d) EC50 = 62.4 mg/kg dw
Avena sativa: (14 d) EC50 = 30.3 mg/kg dw
Executive summary:

The effects on seedling emergence and seedling growth of p-chloro-m-cresol were investigated according to OECD Guideline 208 (Draft Document March 2005) by Buetzler & Meinerling (2007). Two dicotyledons (Brassica napus and Glycine max) and one monocotyledon (Avena sativa) were exposed to concentrations of 0.96, 2.40, 6.00, 15.0, 37.5, 93.8 and 234.4 mg test item/kg soil dry weight (Brassica napus), 2.40, 6.00, 15.0, 37.5, 93.8 and 234.4 mg/kg soil dry weight (Glycine max) and 15.0, 37.5, 93.8 and 234.4 mg/kg soil dry weight (Avena sativa), a water control and a solvent (acetone) control. The exposure duration was 14 days. Test parameters were fresh weight, germination, mortality, phytotoxicity and growth stage. The most sensitive species in terms of fresh weight were Brassica napus and Avena sativa with EC50 values of 27.7 mg test item/kg soil dry weight and 30.3 mg/kg soil, respectively. The EC50 for Glycine max was determined to be 62.4 mg/kg soil. The only statistically significant mortality was observed for Brassica napus at 93.8 mg/kg soil dry weight (45 %). The other mortality counts for Glycine max and Avena sativa turned out to be not statistically significant. The germination rate was significantly reduced for Brassica napus at 93.8 mg/kg soil and at higher concentrations and for Glycine max and Avena sativa at 234.4 mg/kg soil. Phytotoxic effects observed on all plant species were chlorosis, necrosis and growth reduction. All validity criteria of the study were fulfilled. The EC50 value for the most sensitive species Brassica napus was 27.7 mg/kg soil dry weight.

Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to analogue justification document provided in IUCLID section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Species:
Avena sativa
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Avena sativa
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Brassica rapa
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Brassica rapa
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Brassica rapa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight and number of flowers
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Avena sativa
Duration:
46 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh and dry weight of inflorescences
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Avena sativa
Duration:
46 d
Dose descriptor:
NOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh and dry weight, number of inflorescences
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Avena sativa
Duration:
46 d
Dose descriptor:
LOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh and dry weight of inflorescences
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Brassica rapa
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot dry weight and fresh and dry weight of inflorescences
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Brassica rapa
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight and number of inflorescences
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Brassica rapa
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot dry weight, fresh and dry weight of inflorescences
Remarks on result:
other: source, CAS 59-50-7, key rel 1, Förster 2015
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
54.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks:
The result was normalized and converted to a standard soil (3.4% organic carbon + Fom 1.734%)
Remarks on result:
other: source, CAS 59-50-7, rel 2, Buetzler&Meinerling 2007
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
27.7 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks:
95 % CI: 25.3 - 27.7 mg/kg soil dw
Remarks on result:
other: source, CAS 59-50-7, rel 2, Buetzler&Meinerling 2007
Species:
Glycine max (G. soja)
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
62.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks:
95 % CI: 2.65 - 30536 mg/kg soil dw
Remarks on result:
other: source, CAS 59-50-7, rel 2, Buetzler&Meinerling 2007
Species:
Glycine max (G. soja)
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks on result:
other: source, CAS 59-50-7, rel 2, Buetzler&Meinerling 2007
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
30.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks:
95 % CI: 27.7 - 33.2 mg/kg soil dw
Remarks on result:
other: source, CAS 59-50-7, rel 2, Buetzler&Meinerling 2007
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
< 15 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fresh weight
Remarks on result:
other: source, CAS 59-50-7, rel 2, Buetzler&Meinerling 2007

Description of key information

NOECoverall(46 d): 100 mg/kg soil dw, read-across

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:
100 mg/kg soil dw

Additional information

No study investigating the toxicity of sodium p-chloro-m-cresolate (CAS 15733-22-9) to terrestrial plants is available. Therefore, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across to the source substance (p-chloro-m-cresol, CAS 59-50-7) which is the main transformation product of the target substance sodium p-chloro-m-cresolate (CAS 15733-22-9) is applied.

The source substance p-chloro-m-cresol is the common compound in this analogue approach and is solely responsible for the (absence of) effects. Thus, the source substance is considered a suitable representative for the evaluation of the toxicity of the target substance to terrestrial organisms. The read-across approach is justified in detail within the analogue justification in IUCLID section 13.

One key (Förster 2015) and one supporting study (Buetzler & Meinerling 2007) are available investigating the toxicity of the source substance p-chloro-m-cresol (CAS 59-50-7) to terrestrial plants. The key study was conducted according to ISO 22030 (GLP). Seeds of Brassica rapa were exposed for 35 d whereas seeds of Avena sativa were exposed for 46 d to different concentrations of the test item. The study was conducted in a growth chamber under controlled test conditions. The test substance completely inhibited the emergence of A. sativa and B. rapa at a concentration of 400 mg/kg soil dw. At 200 mg/kg soil dw the emergence of B. rapa was still strongly retarded. Reductions in shoot weight and in the weight of inflorescences and seed pods were seen at concentration of 200 mg/kg soil dw. No adverse effects were observed at test concentrations up to 100 mg/kg soil dw. The overall lowest NOEC was 100 mg/kg soil dw.

The effects on seedling emergence and seedling growth of p-chloro-m-cresol in the supporting study were investigated according to OECD Guideline 208 (Draft Document March 2005) by Buetzler & Meinerling (2007). Two dicotyledons (Brassica napus and Glycine max) and one monocotyledon (Avena sativa) were exposed to concentrations of 0.96, 2.40, 6.00, 15.0, 37.5, 93.8 and 234.4 mg test item/kg soil dry weight (Brassica napus), 2.40, 6.00, 15.0, 37.5, 93.8 and 234.4 mg/kg soil dry weight (Glycine max) and 15.0, 37.5, 93.8 and 234.4 mg/kg soil dry weight (Avena sativa), a water control and a solvent (acetone) control. The exposure duration was 14 days. Test parameters were fresh weight, germination, mortality, phytotoxicity and growth stage. The most sensitive species in terms of fresh weight were Brassica napus and Avena sativa with EC50 values of 27.7 mg test item/kg soil dry weight and 30.3 mg/kg soil, respectively. The EC50 for Glycine max was determined to be 62.4 mg/kg soil. The only statistically significant mortality was observed for Brassica napus at 93.8 mg/kg soil dry weight (45 %). The other mortality counts for Glycine max and Avena sativa turned out to be not statistically significant. The germination rate was significantly reduced for Brassica napus at 93.8 mg/kg soil and at higher concentrations and for Glycine max and Avena sativa at 234.4 mg/kg soil. Phytotoxic effects observed on all plant species were chlorosis, necrosis and growth reduction. All validity criteria of the study were fulfilled. The EC50 value for the most sensitive species Brassica napus was 27.7 mg/kg soil dry weight.

Based on the available results from a structurally similar source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which is the main transformation product of the target substance and is characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that sodium p-chloro-m-cresolate has effects in the same range as the target substance.