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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:
2018-11-28 to 2019-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 208 (Terrestrial Plants, Growth Test)
Version / remarks:
July 2006
Deviations:
yes
Remarks:
The room temperature and relative humidity were recorded continuously throughout the test with a data logger and a hygro-thermograph, respectively, due to technical reasons which is a deviation from the study plan.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item Dissolvine E-Fe-13
Batch number CFC 7539
CAS No. 15708-41-5
Chemical name Ethylenediaminetetraacetic acid, ferric-sodium complex
Assay Content (certified) 100 % (calculated)
Iron Content (certified) 13.3 % (SMA 347.05)
Water solubility ca. 90 g/L
pH (certified) 4.9 (1% w/v solution)
Appearance Crystals (=solid), yellow-green
Stability under Not specified
test conditions
Expiry date 2021-05-14
Recommended storage Well closed and stored at room temperature
Analytical monitoring:
yes
Details on sampling:
Determination of the test item
The concentration of the test item EDTA-FeNa was confirmed by analytical verification of the stock solutions and control. The samples were analysed with a photometer method, implemented under non-GLP and documented finally in the GLP raw data.
Vehicle:
yes
Remarks:
Quartz sand
Details on preparation and application of test substrate:
The control and test item treatments were applied once at the start of the exposure.
A stock solution was prepared in demineralised water, diluted to various concentrations and incorporated into the soil. All concentrations of the test item were related to the dry weight of the soil. The maximum water holding capacity (MWHC) and the dry weight of the soil were determined one and two days prior to application, respectively. The test item solutions and additional demineralised water were added to the soil to adjust the humidity of the soil to a moisture of 45 % of the MWHC were added to the soil. Subsequently, the soil was mixed carefully with a mixer (2-3 min) to ensure a homogeneous distribution.
The treated soil was transferred to the test container and the seeds were sown.

Application Conditions of the Soil
Application date 2018-11-28 2019-01-22
Dry Weight (DW) of the soil before application [g/100 g soil WW] 96.2 96.1
Soil amount per batch [kg DW] 7.80* / 5.4 5.5
[kg WW] 8.11 5.72
Maximum water holding capacity (MWHC) of the soil [g/100 g soil DW] 37.8 36.5
Added water amount per application to reach 45 % of MWHC [g/ 100 g DW] 13.06 12.37
Soil amount (with 45 % of MWHC) per replicate [kg] 0.70 0.70
* Due to large soil amounts per concentration, several batches of 7.80 kg DW were prepared per concentration and control and applied separately.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
Common name: Oats
Plant family: Poaceae
Variety: KWS Contender
Source of seed: KWS Lochow GmbH, Ferdinand-von-Lochow-Straße 5, 29303 Bergen, Germany
Prior seed treatment / Sterilization: No
Seed storage: The seeds were stored dark at the test facility at room temperature (22 +/- 2 °C) and protected from moisture until use.
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Common name: Rape
Plant family: Brassicaceae
Variety: Sherlock
Source of seed: KWS Saat SE, Grimsehlstraße 31, 37574 Einbeck, Germany
Prior seed treatment / Sterilization: No
Seed storage: The seeds were stored dark at the test facility at room temperature (22 +/- 2 °C) and protected from moisture until use.
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Common name: Soybean
Plant family: Fabaceae
Variety: Obelix
Source of seed: Delley Samen und Pflanzen AG, Schloss Delley 40, route de Portalban, CH-1567 Delley, Switzerland
Prior seed treatment / Sterilization: No
Seed storage: The seeds were stored dark at the test facility at room temperature (22 +/- 2 °C) and protected from moisture until use.
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
other: 2:1 Mixture of natural soil LUFA 2.2 (loamy sand (DIN classification)) and quartz sand
Limit test:
no
Total exposure duration:
21 d
Test temperature:
nominal: 22 +/- 10 °C; actual: 13 - 23 °C
pH:
5.36 ± 0.04 (soil)
Moisture:
relative humidity:
nominal: 70 +/- 30 %; 33 - 100 %
Details on test conditions:
TEST SYSTEM
- Testing facility: Noack Laboratorien GmbH
- Test container (type, material, size): Non-porous plastic containers (standard flower pots) with a diameter of ca. 12 cm were used. The pots were labelled with the study number and all necessary additional information to ensure unique identification.
- Amount of soil: 700 g per test container
- Method of seeding: Test containers were filled each with the same amount of treated soil (see application technique) and seeding holes were made with a seedling pistil. One seed was given into each hole and covered with soil. During cultivation the test containers were bottom watered and fertilized as necessary.
- No. of seeds per container: Oats, rape: 5; Soybean: 3
- No. of replicates per treatment group: 8
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
A 2:1 mixture of natural soil LUFA 2.2 (batch number: Sp2.23618, loamy sand (DIN classification)) and quartz sand (12a) was used.
Grain size: <= 2 mm
Clay (< 0.002 mm): 6.0 %
Silt (0.002 - 0.063 mm): 9.1 %
Sand (0.063 - 2 mm): 84.9 %
Carbon content: 0.986 %
pH-value: 5.36 ± 0.04

NUTRIENT MEDIUM (if used)
HAKAPHOS® SOFT SPEZIAL 16+8+22(+3)
For watering a 0.05 ‰ solution was used
Origin: Grünes Landhaus, St. Godehard-Str. 23, 31139 Hildesheim, Germany

GROWTH CONDITIONS
- Photoperiod: 16/8
- Light source: Special-fluorescent tube Fluora and Daylight (Osram)
- Light intensity and quality: nominal: > 3500 lx; actual: 4570 ± 491 / 4444 ± 396
- Temperatures: nominal: 22 +/- 10 °C; 13 - 23 °C
- Relative humidity (%): nominal: 70 +/- 30 %; actual: 33 - 100 %
- Watering regime and schedules: Bottom watering of the test containers with fertilized tap water as needed.
- Interval of applications: once at experimental starting
- Method of application: soil incorporation

ACCLIMATION PERIOD: None

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

- Phytotoxicity rating system (if used):
During the 21 day observation period (from day of application) the plants were observed on day 7, 14 and 21, for number of emerged seedlings, number of dead plants and visual phytotoxic effects.
The rating of the treated plants was done in relation to the untreated control plants. Observations included all variations, either inhibitory or stimulatory, between the treated replicates and the untreated controls. Such variations may be phytotoxic symptoms (e.g. chlorosis, necrosis, wilting), effects of growth and development rates. 
Observation Parameters
(1) Deformation of leaves (curling / twisting / sticking / …)
(2) Shoot deformations (curling / bending / …)
(3) Stunted growth
(4) Necrosis
(5) Chlorosis
(6) Lighter green
(7) Darker green
(8) Loss of colour
(9) Discoloration of leaves
(10) Wilting
(E) Dead plant (Number of dead plants)

Assessment of the Effects
- = no effects observed / normal growth
+/- = slight effects
+ = medium effects
++ = strong effects 


VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Oats, soybean: 3; Rape: 2.5
- Range finding study

The preliminary study was performed under the same laboratory conditions as specified for the definitive study with 4 replicates per concentration and control. Test duration was 21 days. At test end shoot fresh weight and emergence were determined.

Inhibition of Shoot Fresh Weight
Test concentration
[mg/kg DW] Inhibition [%]
Oats Rape Soybean
10 -1 -4 -7
100 -6 6 6
1000 -1 79 -

Inhibition of Rate of Emergence
Test concentration
[mg/kg DW] Inhibition [%]
Oats Rape Soybean
10 5 0 11
100 5 10 23
1000 10 75 100
Negative values = promoted growth
DW = soil dry weight
- not emerged

Nominal and measured concentrations:
Nominal: Test item = 100% active ingredient
Oats, soybean: 1000 - 333 - 111 - 37.0 - 12.3 - 4.12 mg/kg DW
Rape: 1000 - 400 - 160 - 64 - 25.6 mg/kg DW
Reference substance (positive control):
no
Key result
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
333 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Key result
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
111 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Key result
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
160 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
714 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Details on results:
The percentage of inhibition of shoot height, shoot fresh weight and rate of emergence is given below. Test item-related statistically significant inhibitions are given in bold. The tabulated results represent rounded mean values calculated on the exact raw data.

Oats: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test concentration Shoot Inhibition Shoot fresh Inhibition Rate of Inhibition
[mg/kg DW] height 1) weight emergence 1)
[cm] [%] [mg] [%] [%] [%]

Control 40.2 - 1333 - 100 -
4.12 39.5 2 1336 0 93 7
12.3 37.5 7 1287 3 95 5
37.0 38.2 5 1338 0 90 10
111 38.7 4 1287 3 95 5
333 38.3 5 1317 1 98 2
1000 36.9 8 1070 20 98 2


Rape: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test concentration Shoot Inhibition Shoot fresh Inhibition Rate of Inhibition
[mg/kg DW] height 2) weight emergence 2)
[cm] [%] [mg] [%] [%] [%]

Control 14.7 - 1235 - 93 -
25.6 14.4 2 1121 9 95 -2
64 14.6 1 1071 13 95 -2
160 14.5 1 1035 16 90 3
400 11.0 25 658 47 85 9
1000 8.1 45 326 74 40 57


Soybean: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test concentration Shoot Inhibition Shoot fresh Inhibition Rate of Inhibition
[mg/kg DW] height 1) weight emergence 1)
[cm] [%] [mg] [%] [%] [%]
Control 22.1 - 2815 - 96 -
4.12 22.8 -3 2885 -2 100 -4
12.3 21.4 3 2638 6 100 -4
37.0 21.8 1 2619 7 96 0
111 20.8 6 2377 16 96 0
333 19.7 11 2202 22 83 14
1000 6.2 72 762 73 8 92
Negative values = Promoted growth
bold = Statistically significant inhibition
1) Normality test failed
2) Normality test and Equal Variance test failed
Reported statistics and error estimates:
Percentage of inhibition
The percentage of inhibition of biomass growth (shoot height, shoot fresh weight) and rate of emergence were calculated in relation to the control. All dead plants were excluded from the calculation of shoot height and fresh weight (above-ground biomass).

NOEC / LOEC of biomass growth / emergence
One Way Analysis of Variance (ANOVA) was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance, a Normality test (Shapiro-Wilk) and an Equal Variance test (Brown-Forsythe) were done first. P-values for both, Normality and Equal Variance test, are 0.05. The alpha-value for ANOVA (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.

Failure of the normality test and / or the equal variance test can be caused by extremely homogeneous emergence and growth patterns as opposed to higher variances in other treatments. Due to the high and even number of replicates in the control and treatment groups, the failure had no influence on the robustness of the calculations.

EC-values and confidence intervals of biomass growth
EC25- and EC50- values were calculated by sigmoidal dose-response regression using GraphPadPrism. Four parameter were used for dose-response fitting. Calculation of the confidence intervals for EC-values was carried out using standard procedures provided by GraphPadPrism.
Validity criteria fulfilled:
yes
Conclusions:
Potential toxic effects of the test item were assessed on day 7, 14 and 21 by visual observations (number of emerged seedlings and phytotoxic effects) and at test end (day 21) by shoot height and shoot fresh weight determination.
The results of the control for all plant species met the required validity criteria. Therefore, the test is considered to be valid.

Shoot Height: NOEC, LOEC, EC25- and EC50-Values with Confidence Range (p)
Species NOEC
[mg/kg DW] LOEC
[mg/kg DW] EC25
[mg/kg DW] p = 95%
[mg/kg DW] EC50
[mg/kg DW] p = 95%
[mg/kg DW]
Oats 333 1000 > 1000 – > 1000 –
Rape 160 400 483 373 - 598 > 1000 –
Soybean 111 333 480 410 - 564 714 621 - 835


Shoot Fresh Weight: NOEC, LOEC, EC25- and EC50-Values with Confidence Range (p)
Species NOEC
[mg/kg DW] LOEC
[mg/kg DW] EC25
[mg/kg DW] p = 95%
[mg/kg DW] EC50
[mg/kg DW] p = 95%
[mg/kg DW]
Oats 333 1000 > 1000 – > 1000 –
Rape 160 400 236 143 - 327 430 328 - 619
Soybean 37.0 111 320 241 - 431 692 569 - 849


Number of Emerged Seedlings: NOEC, LOEC, EC25- and EC50-Values with Confidence Range (p)
Species NOEC
[mg/kg DW] LOEC
[mg/kg DW] EC25
[mg/kg DW] p = 95%
[mg/kg DW] EC50
[mg/kg DW] p = 95%
[mg/kg DW]
Oats ≥ 1000 > 1000 > 1000 – > 1000 –
Rape 400 1000 583 422 - 756 877 660 - > 1000
Soybean 333 1000 404 342 - 492 537 454 - 655
-) Not determinable
Executive summary:

Summary

The phytotoxicity of the test itemDissolvineE-Fe-13(batch number:CFC7539) to three terrestrial plant species was determined according to OECD 208 (July 2006) atthe test facilityin seedling emergence and seedling growth tests over a period of 21 days, from2018-11-28 to 2019-02-12.

Test Design

Test systems comprised one monocotyledon (oats, Poaceae), and two dicotyledons (rape, Brassicaceae; soybean, Fabaceae).

 

The test was conducted with various test concentrations as stated below:

Oats, soybean: 1000 - 333 - 111 - 37.0 - 12.3 - 4.12 mg/kg DW (factor 3)

Rape: 1000 - 400 - 160 - 64 - 25.6 mg/kg DW (factor 2.5)

 

The test item was incorporated into the soil in which the seeds were sown. The test containers were bottom watered with nutrient solution as needed.

 

Results

The stock solution was sampled prior to application and subsequently analytically verified. The measured concentration of Dissolvine E-Fe-13 was 91 and 89 % indicating the correct preparation of the stock solutions.

 

Potential toxic effects of the test item were assessed on day 7, 14 and 21 by visual observations (number of emerged seedlings and phytotoxic effects) andat test end by shoot height and shoot fresh weight determination.

  

The results of the control for all plant species met the required validity criteria. Therefore, the tests are considered to be valid.

 

 

Shoot Height: NOEC, LOEC, EC25- and EC50-Values with Confidence Range (p)

Species

NOEC
[mg/kg DW]

LOEC
[mg/kg DW]

EC25
[mg/kg DW]

p = 95%
[mg/kg DW]

EC50
[mg/kg DW]

p = 95%
[mg/kg DW]

Oats

333

1000

> 1000

> 1000

Rape

160

400

483

373 - 598

> 1000

Soybean

111

333

480

410 - 564

714

621 - 835

 

Shoot Fresh Weight: NOEC, LOEC, EC25- and EC50-Values with Confidence Range (p)

Species

NOEC
[mg/kg DW]

LOEC
[mg/kg DW]

EC25
[mg/kg DW]

p = 95%
[mg/kg DW]

EC50
[mg/kg DW]

p = 95%
[mg/kg DW]

Oats

333

1000

> 1000

> 1000

Rape

160

400

236

143 - 327

430

328 - 619

Soybean

37.0

111

320

241 - 431

692

569 - 849

Number of Emerged Seedlings: NOEC, LOEC, EC25- and EC50-Values with Confidence Range (p)

Species

NOEC
[mg/kg DW]

LOEC
[mg/kg DW]

EC25
[mg/kg DW]

p = 95%
[mg/kg DW]

EC50
[mg/kg DW]

p = 95%
[mg/kg DW]

Oats

≥ 1000

> 1000

> 1000

> 1000

Rape

400

1000

583

422 - 756

877

660 - > 1000

Soybean

333

1000

404

342 - 492

537

454 - 655

-) Not determinable

Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Category justification document is attached in chapter 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Species:
Oryza sativa
Key result
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
111 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Remarks on result:
other: EDTA-FeNa
Species:
other: Nicotiana tabacum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 84 - < 420 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: EDTA-NA2H2
Species:
Phaseolus vulgaris
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 58.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: EDTA
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
333 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Remarks on result:
other: EDTA-FeNa
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
160 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: growth
Remarks:
shoot height
Remarks on result:
other: EDTA-FeNa
Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 227 (Terrestrial Plant Test: Vegetative Vigour Test)
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Details on preparation and application of test substrate:
A silty–loamy sand agricultural soil, USDA (Soil Survey Manual), was collected from 0 to 30 cm surface layer of the Melaten field in Aachen, Germany. The soil was air-dried at room temperature, sieved through a 2-mm sieve and characterised as follows:

pH: 6.8
organic matter: 3.5 %
soil fractions
sand: 49.4 %
Clay: 42.1 %
Silt: 8.5 %
Species:
other: Nicotiana tabacum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Tobacco plants (N. tabacum SR-1) were used for the experiment.
Test type:
vegetative vigour test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
21 d
Remarks:
approximately 3 weeks
Post exposure observation period:
no data available
Test temperature:
25/20 °C light/dark temperature regime
pH:
6.40 - 6.83
Moisture:
60% relative humidity
Details on test conditions:
16 h light period (light intensity of 320 µmol/m2 * s)
Nominal and measured concentrations:
Nominal: 0.125,0.25, 1.25, 62.5, 125, and 250 mmol/kg for Na2EDTA
Reference substance (positive control):
not specified
Key result
Species:
other: Nicotiana tabacum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 84 - < 420 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Key result
Species:
other: Nicotiana tabacum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.25 - < 1.25 other: mmol/kg
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Details on results:
EDTA did not show any adverse effects at a concentration of < 0.125 mmol/kg. At 1.25 mmol/kg of EDTA the leaves showed necrotic lesions and the dry weight was negligible. At a concentration of 0.25 mmol/kg EDTA the dry weight is only slightly lower but the standard deviation is higher in comparison to the 0.125 mmol/kg application.
Results with reference substance (positive control):
no data available
Reported statistics and error estimates:
no data availble
Validity criteria fulfilled:
not specified
Conclusions:
Based on the results of this vegetative vigour study EDTA had an inhibitory effect on growth of Nicotiana tabacum at rate of 1.25 mmol/kg soil (equivalent to 420 mg/kg). The NOEC will be determined to be 0.25 mmol/kg soil (equivalent to 84 mg/kg soil).
Executive summary:

The effects of EDTA (Na2EDTA) on vegetative vigour of plants following application were examined in a laboratory test on Nicotiana tabacum. The test item was applied at a dose rate of up to 250 mmol/kg and compared to an untreated control.

According to the results, the test item had an inhibitory effect on growth of the tested species at rates of 1.25 mmol/kg soil (equivalent to 420 mg/kg) and thus determined to be the overall LOEC for this species.

The overall NOEC was determined to be 0.25 mmol/kg soil (equivalent to 84 mg/kg).

Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
seedlings of Phaseolus vulgaris were exposed to EDTA for 14 days
GLP compliance:
no
Analytical monitoring:
not specified
Vehicle:
no
Species:
Phaseolus vulgaris
Plant group:
Dicotyledonae (dicots)
Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
other: seedlings grown in polystyrene squares
Limit test:
no
Total exposure duration:
14 d
Details on test conditions:
Bean seeds received a cold treatment (+ 4 °C) for 3 days to break dormancy and to synchronize germination. They were transferred to a growth chamber to germinate between two layers of water-soaked rock wool for 4 days. Subsequently seed coats were removed and seedlings with a root length of approximately 1.5 cm were grown in 3-mm thick polystyrene squares by fixing the roots through 5-mm holes (9 plants/treatment). The polystyrene was floated on 3 L of aerated full strengths Hoagland's solution (pH 5.0) in 3.5 L polyethylene beakers; Fe was added as FeSO4 * 7H2O. To keep Fe in solution, it was complexed with EDTA (Fe/EDTA ratio 2/1). Nutrient solution was replaced every 2 days; pH was monitored daily. Plants were grown under controlled conditions in a 12-h photoperiod at 65 % relative humidity and day/night temperatures of 22°C. Light was supplied by cool white fluorescent lamps (L = 140W/20SA; Osram, Augsburg, Germany) at a photo synthetic photon flux density of 165 µmol/m2 * s at the leaf level. Final concentrations of 0 (= untreated), 50 and 200 µM EDTA in Hoagland's medium, equivalent to 14.6 and 58.4 mg/kg, respectively. Plants were harvested 14 days after the start of treatment, and morphological parameters (root weight, shoot length, leaf weight) were measured.
Nominal and measured concentrations:
Nominal: 14.6 and 58.4 mg/L
Reference substance (positive control):
no
Key result
Species:
Phaseolus vulgaris
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 58.4 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence

Addition of EDTA alone to the growth medium increased EDTA chelation of most divalent cations, resulting in decreased plant uptake of Fe, Mn and Zn. EDTA application however, did not induce any antioxidative stress response. Addition of 200 µM EDTA resulted in a free EDTA concentration of 10 µM in solution.Obviously this was too low to induce any phytotoxic effects.

Validity criteria fulfilled:
not applicable
Conclusions:
In a 14 day exposure test EDTA showed no inhibitory effect on seedling emergence and growth of Phaseolus vulgaris up to a concentration of 58.4 mg/kg soil.
Executive summary:

In a 14 day eposure test EDTA showed no inhibitory effect on seedling emergence and growth of Phaseolus vulgaris up to a concentration of 58.4 mg/kg soil.

Description of key information

NOEC(21d) = 111 mg/kg soil dw (nominal) for growth of Glycine max (OECD 208, read across)

Key value for chemical safety assessment

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

Additional information

Since no studies are available investigating the toxicity of Diammonium dihydrogen ethylenediaminetetraacetate (CAS 20824-56-0) to terrestrial plants, the assessment was based on studies conducted with subcategory 1a members Edetic acid (CAS 60-00-4) and Disodium dihydrogen ethylenediaminetetraacetate (CAS 139-33-3) and subcategory 1b member Sodium feredetate (CAS 15708-41 5) in a read across approach. This is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Grouping of substance and read across approach. Further justification for suitability of read across between Subcategory 1 members is given within the category justification attached to chapter 13.


The key study with sodium feredetate was performed according to OECD guideline 208 in compliance with GLP using Avena sativa, Brassica napus and Glycine max as test organism (Klix 2019). Concentrations between 4.12 and 1000 mg/kg soil dw were tested in a 2:1 mixture of Lufa (2.2) soil and quartz sand over an exposure time of 21 days. Seedling emergence and growth were observed. Growth showed to be more susceptible than emergence. The 21d-NOEC values are determined to be >= 333 mg/kg soil dw for Avena sativa, 160 mg/kg soil dw for Brassica napus and 111 mg/kg soil dw for Glycine max based on nominal (analytically verified) concentrations.


The supporting study with Disodium dihydrogen ethylenediaminetetraacetate was performed according to OECD guideline 227 (vegetative vigour test) using Nicotiana tabacum as test organism (Evangelou et al. 2006). Concentrations between 84 and 84000 mg/kg soil dw were tested in natural soil over an exposure time of 21 days. EDTA did not show any adverse effects at a concentration of 84 mg/kg soil dw. At 420 mg/kg soil dw the leaves showed necrotic lesions and the dry weight was only slightly lower, but the standard deviation is higher in comparison to the 84 mg/kg treatment. Thus, the 21d-NOEC value is determined to be >= 84 - < 420 mg/kg soil dw.


In a second supporting study with edetic acid seedling emergence of Phaseolus vulgaris was tested over an exposure time of 14 days. The non-GLP study was performed similar to OECD 208. Bean seeds were transferred to a growth chamber to germinate between two layers of water-soaked rock wool for 4 days. Subsequently seedlings with a root length of 1.5 cm were grown in polystyrene squares. The polystyrene was floated on Hoagland's solution in 3.5 L polyethylene beakers. Final concentrations of 14.6 and 58.4 mg/L were applied. No inhibitory effect on seedling emergence and growth were observed throughout the test. Thus, the 14-NOEC is determined to be >= 84 mg/L.


Being the study of highest quality and showing the highest effects values, the key study and specifically the lowest observed 21d-NOEC of 111 mg/kg soil dw for Glycine max was used to set a suitable NOEC value for the hazard assessment of Diammonium dihydrogen ethylenediaminetetraacetate (CAS 20824-56-0).