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Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Jan 2014 to 26 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
other: ISO 22030
Principles of method if other than guideline:
"This International Standard describes a plant test that includes both acute and chronic endpoints. The test measures emergence, early growth and reproduction of two terrestrial plant species (Avena sativa and a rapid-cycling variety of Brassica rapa are recommended). The test compares responses of plants in a test soil and/or a series of dilutions with a control soil. This test method may also be used for the testing of chemicals by applying various concentrations of the test substance to a standard control soil. Seeds of both plant species are planted in pots containing the soil/soil mixtures and in control pots containing a reference or standard soil. Pots are placed in a temperature- and light-controlled room or growth chamber. They are watered via wicks. After emergence of the plants, emergence rates are determined and plants are thinned out to a specified number. After two weeks, some of the plants are harvested to determine their biomass. After another period of three weeks to four weeks (rapid-cycling Brassica rapa) or five weeks to 6 weeks (Avena sativa), the remaining plants are harvested for measuring additional endpoints characterizing their
reproductive potential. In all cases, the test duration should be sufficient to determine reproductive endpoints (e.g. number or biomass of flowers or seeds or fruit). Typically, 10 seeds are sown in four replicate test pots each. Plants are thinned out to 8 per pot, and four plants each are harvested at day 14 and at the end of the test. If in any pot less than 8 plants have emerged, the number of plants harvested at day 14 shall be reduced such that four plants remain for the final harvest. The relative inhibition in undiluted test soils is determined to assess the suitability of the soil for plants. In addition, based on a dilution series, NOEC, LOEC and ECx values can be calculated from the dose response curves. The latter is required when chemicals are tested."
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Details on sampling:
- Concentrations: 10.0, 31.6, 100, 316, 1000 mg/kg soil dry weight
- Sampling method: Not required - test results are based on nominal concentrations
Vehicle:
no
Details on preparation and application of test substrate:
- For A. sativa, the stock solution was prepared by diluting 12.80 g of cyanoguanidine in 500 ml of deionised water. Test solutions of 250 ml were obtained through dilutions of the stock solution with deionised water.
- For B. rapa, the stock solution was prepared by diluting 8.00 g of cyanoguanidine in 500 ml of deionised water. Test solutions of 200 ml were obtained through dilutions of the stock solution with deionised water
- Test solutions were mixed into the bulk soils for the treatments. Soil was thoroughly mixed by an electric mixer for several minutes.
- Testing of a reference item is not required by the test guideline. Therefore, the test was performed without a reference item.
- Untreated water control
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Oat
- Plant family: Poaceace
- Variety: St Champion
- Source of seed: Saaten-Union GmbH, Isernhagen, Germany
- Prior seed treatment/sterilization: No; the seeds were neither dressed with fungicides nor imbibed with water
- Historical germination of seed (germination of seed lot tested): No
- Seed storage: Not reported
Species:
Brassica rapa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Turnip rape
- Plant family: Brassicaceae
- Variety: Wisconsin fast plants
- Source of seed: Carolina Biological Supply Company, Burlington NC, USA
- Prior seed treatment/sterilization: No; the seeds were neither dressed with fungicides nor imbibed with water
- Historical germination of seed (germination of seed lot tested): No
- Seed storage: Not reported
Test type:
other: Emergence, early growth and reproduction
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
38 d
Remarks:
Avena sativa: 38d; Brassica rapa: 30d
Test temperature:
Mean air temperature in the test room was 24.1° C with a minimum of 19.0 °C and a maximum of 27.3 °C.
pH:
6.0 ± 0.9
Moisture:
6.8 (% of dry weight)
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): Rectangular polystyrene beakers, 18.3 cm by 13.6 cm large and 6.4 cm high (Kastelplast GmbH, Mainz, Germany) were used as test pots and also as water reservoirs for the test pots
- Amount of soil: approximately 900 g
- No. of seeds per container: 10 of each species per pot
- No. of plants (retained after thinning): 8
- No. of replicates per treatment group: 4 pots per tst species and test condition
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Natural field soil, Lufa standard soil type 2.3, Germany, Rhineland-Palatinate, Offenbach, "rechts der Landauer Str.", field number 826/7
- Pesticide use history at the collection site: Fallow; no fertilizer or pesticides during the last four years
- Sampling depth (cm): 20 cm
- Soil texture (if natural soil): sandy loam
- pH = 6.0 ± 0.9 (in 0.01 M CaCl2)
- Organic carbon (%): 0.67 ± 0.05
- Maximum water holding capacity (g/100g): 36.2 ± 2.0
- Cation exchange capacity (meq/100 g): 6.9 ± 1.0
- Storage (conditions before test): ambient

GROWTH CONDITIONS
- Photoperiod: 16 h per day (8 hours darkness)
- Light source: high pressure metal halide lamps (type Master SON-T-Agro, Philips GmbH, Hamburg, Germany)
- Light intensity and quality: Light intensity ranged from 12380 to 17820 Lux
- Day/night temperatures: Mean air temperature in the test room was 24.1 °C with minimum 19.0 °C and maximum of 27.3 °C
- Relative humidity (%): Mean relative air humidity was 34.2 % with minimum of 19.5 % and maximum of 55.1 %
- Air exchange rate: Not reported; however, all pots were randomly distributed in an air conditioned room, pots were rearranged randomly once per week
- Watering regime and schedules: Water or nutrient solution was added to the water reservoirs as needed
- Water source/type: Each pot was placed on a separate water reservoir
- Volume applied: Pots were equipped with glass fibre wicks with a diameter of 10 mm (Frenzelit lsoGias-Schnur, Muhlberger, Wiesbaden, Germany) to connect the soil in the pot with the water reservoir

VEHICLE CONTROL PERFORMED: yes - water control

TEST CONCENTRATIONS
- Concentrations were defined by the study director in consultation with the sponsor and according to the results of a previously performed non-GLP range-finding test according to guideline ISO 11269-2
Nominal and measured concentrations:
10.0, 31.6, 100, 316 and 1000 mg/kg soil dry weight, water control (all nominal)
Reference substance (positive control):
no
Remarks:
Testing of a reference item is not required by the test guideline. Therefore, the test was performed without a reference item.
Species:
Avena sativa
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 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.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
shoot length
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:
growth
Remarks:
shoot fresh weight
Species:
Avena sativa
Duration:
38 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
shoot length
Species:
Avena sativa
Duration:
38 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
shoot fresh weight
Species:
Avena sativa
Duration:
38 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
shoot dry weight
Species:
Avena sativa
Duration:
38 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction: number of inflorescences
Species:
Avena sativa
Duration:
38 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction: fresh weight of inflorescences
Species:
Avena sativa
Duration:
38 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction: dry weight of inflorescences
Species:
Brassica rapa
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
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:
growth
Remarks:
shoot length
Species:
Brassica rapa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
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: number of flowers
Species:
Brassica rapa
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
shoot length
Species:
Brassica rapa
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
shoot fresh weight
Species:
Brassica rapa
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
shoot dry weight
Species:
Brassica rapa
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction: number of seed pods
Species:
Brassica rapa
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction: fresh weight of seed pods
Species:
Brassica rapa
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction: dry weight of seed pods
Reported statistics and error estimates:
- Reported by arithmetic means per pot (shoot fresh weight, number of flowers, number of inflorescences, number of seed pods), or on the
basis of the sum per pot (number of seedlings, fresh and dry weight of A. sativa inflorescences and fresh and dry weight data B. rapa seed pods).
- Normal distribution was tested by the Kolmogorov-Smirnov test, homogeneity of variances by Cochran's test.
- NOEC was either determined by applying Williams' multiple sequential t-test (p<0.05, one sided smaller) or, if homogeneity of variance was denied, by using the Welch t-test for inhomogeneous variances with Bonferroni adjustment (p<0.05, one sided smaller)
- Statistical evaluations were performed using the ToxRat software Version 2.10
Validity criteria fulfilled:
yes
Conclusions:
The validity criteria for controls as defined by the guideline have been fulfilled:
- Emergence rate in untreated control soil was 95 % for A.sativa and 90 % for B.rapa (guideline criterion: ≥ 75 %)
- Healthy plants developed and did not etiolate and flowers appeared during first five weeks for A. sativa or three weeks for B. rapa
- Not more than one emerged plant died during the test
The authors conclude that cyanoguanidine (dicyandiamide) caused adverse effects on growth and development of Avena sativa and Brassica rapa seedlings at concentrations of ≥ 100 mg/kg soil dry weight. The overall NOEC from the chronic plant test is 31.6 mg/kg soil dry weight.
Executive summary:

The effect of cyanoguanidine (dicyandiamide) on the emergence, early growth and reproduction of the monocot Avena savtiva and the dicot Brassica rapa rapa crops was studied at nominal concentrations of 10.0, 31.6, 100, 316 and 1000 mg/kg soil dry weight. The growth medium used in the growth and reproduction test was natural soil (sandy loam, pH 6.0, organic carbon 0.67 %, CEC = 6.9). On day 7 the surviving plants per pot were recorded and thinned to eight plants per pot.

Emergence of A. sativa was not affected by the treatment with cyanoguanidine (dicyandiamide) whereas emergence of B. rapa was affected at the highest concentration of 1000 mg/kg soil dw. The NOEC based on seed germination in the more sensitive species (B. rapa) was 316 mg/kg soil dw.

Growth of A. sativa after 14 days was significantly affected at 100 mg/kg soil dw (in terms of shoot length) or at 316 mg/kg soil dw (in terms of shoot fresh weight). In B. rapa, growth after 14 days was significantly affected at 316 mg/kg soil dw (in terms of shoot length) or at 100 mg/kg soil dw (in terms of shoot fresh weight). The NOEC based on growth after 14 days was 31.6 mg/kg soil dw in both species.

Growth of A. sativa upon final harvest (38 days) was significantly affected at 316 mg/kg soil dw (in terms of shoot fresh weight) whereas there was no effect on shoot fresh weight up to the highest concentration. In B. rapa, growth upon final harvest (30 days) was significantly affected at 1000 mg/kg soil dw (in terms of shoot length) or at 100 mg/kg soil dw (in terms of shoot fresh weight). Therefore, the NOEC based on the most sensitive growth parameter was 31.6 mg/kg soil dw in the more sensitive species (B. rapa).

Reproduction of A. sativa was significantly affected at 100 mg/kg soil dw (in terms of number, fresh weight, and dry weight of inflorescences upon final harvest). In B. rapa, reproduction was significantly affected at 316 mg/kg soil dw (in terms of number of seed pods) or at 100 mg/kg soil dw (in terms of both fresh and dry weight of seed pods). Therefore, the NOEC based on reproduction was 31.6 mg/kg soil dw in both species.

No abnormalities were noted.

Results Synopsis

Chronic toxicity plants: Emergence, growth, reproduction

Monocot

Avena sativa (oat)

NOEC: 31.6 mg/kg soil dw

Most sensitive parameter: Number, fresh and dry weight of inflorescences

Dicot

Brassica rapa rapa (turnip rape)

NOEC: 31.6 mg/kg soil dw

Most sensitive parameter: Fresh and dry weight of seed pods

This toxicity study is classified as acceptable and satisfies the guideline requirement for a chronic plant toxicity study.

Description of key information

A study on the chronic toxicity of cyanoguanidine (dicyandiamide) to plants, investigating effects on the endpoints emergence, growth, and reproduction, was conducted. The method followed the test guideline ISO 22030 (Soil quality — Biological methods —

Chronic toxicity in higher plants). Adverse effects were observed, resulting in an overall NOEC (based on reproduction parameters) of 31.6 mg/kg soil dw.

Key value for chemical safety assessment

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

Additional information