Iron(3+) ion dipotassium 2-({2-[bis(carboxylatomethyl)amino]ethyl} (carboxylatomethyl)amino)acetate; hydrate

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The results as observed/predicted for EDTA-FeNa are read across to EDTA-Fe(OH)K2 as in diluted form in the environment both EDTA complexes will have a similar structure and related fate.

2. ANALOGUE APPROACH JUSTIFICATION
It is therefore considered justified to use data/predictions as observed for EDTA-FeNa also for EDTA-Fe(OH)K2.

3. ANALOGUE APPROACH JUSTIFICATION
It is considered justified to use the results as observed/predicted for EDTA-FeNa for read across to EDTA-Fe(OH)K2 because in diluted form in the environment both EDTA complexes will have a similar structure and related fate.

4. DATA MATRIX
See chapter 13 for Read across document

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984 version

Deviations:

no

Principles of method if other than guideline:

No

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Remarks:

Substance has a high water solubility

Details on preparation and application of test substrate:

The respective test item amount was weighed out for a stock solution (nominal: 2220 mg/100 mL, actual: 2220 mg/100 mL) and dispersed in demineralised water. The stock solution was further diluted to the respective application solutions. The application solution and additional demineralised water were added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 555 g SDW were filled into each test vessel.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Test organism Eisenia fetida (Annelida, Lumbricidae)
Reason for the selection Eisenia fetida is suitable for this kind of study and is one of the recommended species according to the guideline.
Source Breeding stock culture maintained at the test facility
Breeding Organisms of the species Eisenia fetida are bred at the test facility in covered plastic vessels containing potting compost.
Breeding conditions Temperature of 20 ± 2°C in the dark
Feeding During breeding, the earthworms are fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food is provided depending on the feeding rate and the density of the earthworm population in the vessels.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

18 - 23 °C
The room temperature increased to 23 °C for about 6 hours. Since adaptation of the soil to room temperature is very slow, this deviation is considered to have no impact on quality and integrity of the study.

pH:

Control: Day 0 = 6.12; Day 14 = 5.74
316 mg/kg dw: Day 0 = 6.24; Day 14 = 5.73
Range: 5.70 - 6.32

Moisture:

Control: Day 0 = 24.6; Day 14 = 23.7
316 mg/kg dw: Day 0 = 23.3; Day 14 = 22.9
Range: 22.6 - 24.6

Details on test conditions:

The WHCmax, pH-value and the moisture content of the artificial soil were determined prior to the adaptation of the earthworms. At the start and the end of the test, the pH-value and moisture content of the test medium were analysed in the treatments and the control using mixed samples of all replicates.
The live weight of the earthworms was determined individually on the day of application (day 0) and on day 14.
The mean weight change was assessed on day 14 after application.
Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 days after application.

Nominal and measured concentrations:

Nominal: 0, 3.16, 10, 31.6, 100, 316 mg/kg dw

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide (SIGMA-ALDRICH)

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

133 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

129 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Body Weight

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:

other: Mortality, behaviour, pathological symptoms

Reported statistics and error estimates:

LC values The LC10/20/50 values were determined by Probit analysis using linear max. likelihood regression.
For details, please refer to section 12.

EC values The EC10/20/50 values were determined by Analysis of Variance and Test for Lack of Fit for the 3-param. normal CDF.
For details, please refer to section 12.

NOEC, LOEC and statistical The NOEC/LOEC for mortality was determined using Multiple
analyses Sequentially-rejective Fisher Test After Bonferroni-Holm. When running the test, a Qualitative Trend Analysis by Contrasts and Tarone’s Test Procedure were done first.
Statistically significant differences in biomass compared to the control were determined using a Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm. When running the test, Shapiro-Wilk’s Test on Normal Distribution and Levene’s Test on Variance Homogeneity (with Residuals) were done first. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.
For details, please refer to section 12.

1.1  Definitive Test

1.1.1  Mortality

 

A statistically significant mortality was observed in the test item concentrations 100 mg/kg and 316 mg/kg soil dry weight, whereas no mortalities were observed in the control and the test item concentrations 3.16 to 31.6 mg/kg soil dry weight after 14 days of exposure (Table 2).For LC-values please refer to Table 1.

 

Table 2:                Earthworm Mortality in [%] after 7 and 14 Days of Exposure

								Mortality [%]
Test item concentration [mg/kg SDW]	Day 7									Day 14
	1		2		3		4		MV	1	2	3	4	MV
Control	0		0		0		0		0	0	0	0	0	0
3.16	0		0		0		0		0	0	0	0	0	0
10	0		0		0		0		0	0	0	0	0	0
31.6	0		0		10		0		2.50	0	10	10	0	5.00
100	10		10		10		0		7.50	20	20	30	20	22.5
316	80		70		50		60		65.0	100	90	100	90	95

MV = mean value

 

1.1.2  Behaviour and Pathological Symptoms

 

No significant pathological symptoms or changes in the earthworm behaviour were observed in the control as well as in the test item concentrations 3.16 to 31.6 mg/kg soil dry weight after 7 and 14 days of exposure. In the test item concentration 100 mg/kg soil dry weight and 316 mg/kg soil dry weight, significant changes in earthworm behaviour were observed after 7 and 14 days (Table3).

 

Table3:Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure

Test item concentration [mg product/kg SDW]		Effect		Replicate
			1		2		3		4
			Day 7
Control		A)		10/10		10/10		10/10		10/10
3.16		A)		10/10		10/10		10/10		10/10
10		A)		10/10		10/10		10/10		10/10
31.6		A) D) H)		10/10 - -		10/10 - -		9/10 - 1/10		10/10 - -

A) no obvious pathological symptoms             D) spontaneous segmentation and separationH) dead earthworm

 

 

Table 3:       Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure

                    (continued)

Test item concentration [mg product/kg SDW]		Effect		Replicate
			1		2		3		4
			Day 7
100		A) B) C) D) H)		9/10 - - - 1/10		8/10 - - 1/10 1/10		6/10 2/10 1/10   1/10		9/10 1/10 -
316		A) B) C) D) H)		- 2/10 -   8/10		1/10 1/10 - 1/10 7/10		- - 4/10 1/10 5/10		1/10 - 3/10 - 6/10
			Day 14
Control		A)		10/10		10/10		10/10		10/10
3.16		A)		10/10		10/10		10/10		10/10
10		A)		10/10		10/10		10/10		10/10
31.6		A) H)		10/10 -		9/10 1/10		9/10 1/10		10/10 -
100		A) B,D) C,D) H)		6/10 - 2/10 2/10		8/10 - - 2/10		6/10 1/10 - 3/10		8/10 - - 2/10
316		A) H)		- 10/10		1/10 9/10		- 10/10		1/10 9/10

A) no obvious pathological symptoms             

B) no reaction to touching

C) no negative phototactical reaction

D) Spontaneous segmentation and separation

H) dead earthworm

 

 

Live Weight of the Earthworms

 

The individual live weights of the earthworms at test start as well as the mean weights and standard deviations of the replicates of the control and the test item concentration are given in Table 8 toTable 13. The data for individual live weights of the earthworms on day 14 as well as the mean values and the standard deviations of the control and the test item concentration are given in Table 14 to Table19.

During the test, the biomass loss of the earthworms was below 20 % in the control (Table 4).

 

Table4:      Mean Live Weight and Mean Loss of Body Weight

		Mean live weight
Test item  concentration  [mg product/kg SDW]		Test start MV±SD [g/worm]		Test end MV±SD [g/worm]		Inhibition [%]		Statistically significant vs.  Control
Control		0.48 ± 0.03		0.43 ± 0.04		10.4		–
3.16		0.48 ± 0.03		0.43 ± 0.04		10.4		No
10		0.48 ± 0.03		0.44 ± 0.03		8.33		No
31.6		0.48 ± 0.04		0.43 ± 0.05		10.4		No
100		0.47 ± 0.04		0.36 ± 0.10		23.4		Yes
316		0.48 ± 0.04		0.39 ± 0.04		18.8		Yes

MV = Mean value          SD = Standard deviation

 

Physical Data

 

The initial pH of the artificial soil was 5.97 measured on day - 8. Measurements of pH-values and moisture content of the test medium based on mixed samples of all replicates per test item treatment and control are given in Table 5. The environmental conditions throughout the study are summarized in Table 6.

 

Table 5:      pH-Values, Moisture and WHC_maxof the Test Media

Test item concentration  [mg product/kg SDW]	pH-value		Moisture [%] of DW		WHCmax [g /100 g SDW]
	day 0	day 14	day 0	day 14
Control	6.12	5.74	24.6	23.7	48.2
3.16	6.32	5.74	23.9	23.1
10	6.18	5.77	23.5	22.8
31.6	6.19	5.71	23.3	22.7
100	6.29	5.70	23.5	22.6
316	6.24	5.73	23.3	22.9

 

Table 6:      Environmental Conditions

	Room temperature [°C]	Photoperiod [h]	Light intensity [lx]
nominal	20±2	24	400 - 800
actual	18 – 23*		503 ± 56.5

*) The room temperature increased to 23 °C for about 6 hours. Since adaptation of the soil to room temperature is very slow, this deviation is considered to have no impact on quality and integrity of the study.

 

Test of Reference Item

 

The acute toxicity of 2-Chloroacetamide on the earthworm Eisenia fetida fetida (Savigny) was determined over a period of two weeks from 2018-01-26 to 2018-02-09. The LC₅₀value was in the range between 20 and 80 mg/kg SDW as recommended by the guideline (Table7).

 

Table7:         NOEC and LC-Value after 14 d for the Reference Item2-Chloroacetamide

LC-Value / NOEC	Concentration [mg reference item/kg SDW]	95 % - Confidence Interval [mg reference item/kg SDW]
NOEC	30	–
LC50	42.3	n.d.*

  *) not determined due to mathematical reasons (3 reference item concentrations)

Validity criteria fulfilled:

yes

Conclusions:

A valid study performed under GLP conditions according to a standard guideline.

Executive summary:

The acute effects of Dissolvine E-Fe-13 on the earthworm Eisenia fetida were determined according to OECD Guideline 207. The study was conducted under static conditions for 14 days with the test item concentrations 3.16 – 10 – 31.6 – 100 – 316 mg/kg soil dry weight mixed into artificial soil containing 10 % peat. Artificial soil without test item was used as control. 40 test organisms with an individual weight between 0.40 - 0.54 g, divided into four replicates, were tested per test item concentration and control.

A statistically significant mortality was observed in the test item concentrations 100 and 316 mg/kg soil dry weight after 14 days of exposure, whereas no mortalities were observed in the control and the test item concentrations 3.16 to 31.6 mg/kg soil dry weight. Consequently, the NOEC was determined to be 31.6 mg/kg soil dry weight and LOEC was determined to be 100 mg/kg soil dry weight. In terms of body weight loss, theNOEC was determined to be 31.6 mg/kg soil dry weight and LOEC was determined to be 100 mg/kg soil dry weight.

For all LC - and EC_x- values please refer to Table 1. 

All validity criteria of the test guideline were fulfilled.

 

Table1:         Summary of all Effects

Effects	Dissolvine E-Fe-13 [mg/kg soil dry weight]
LOEC Mortality, behaviour, pathological symptoms	100
NOEC Mortality, behaviour, pathological symptoms	31.6
LOEC Change of body weight	100
NOEC Change of body weight	31.6
LC10mortality(confidence interval) LC20mortality(confidence interval) LC50mortality(confidence interval)	56.8 (7.84 - 95.9)                                  76.1 (18.3 – 123) 133 (70.1 - 262)
EC10body weight(confidence interval) EC20body weight(confidence interval) EC50body weight(confidence interval)	51.3 (38.3 – 68.7) 70.4 (52.9 – 93.8) 129 (88.3 – 185)

Description of key information

The ecotoxicity of EDTA-FeK/EDTA-FeNa are considered to be similar to the ecotoxicity of EDTA-Fe(OH)K2 as these substances will be tested in very diluted form in buffered matrices and under these conditions no significant effects of the additional OH or the difference between sodium and potassium are expected. The ecotoxicity test results as observed for EDTA-FeK/ EDTA-FeNa will therefore be read across to EDTA-Fe(OH)K2.

The acute effects of EDTA-FeNa on the earthworm Eisenia fetida were determined according to OECD Guideline 207. The study was conducted under static conditions for 14 days with the test item concentrations 3.16 – 10 – 31.6 – 100 – 316 mg/kg soil dry weight mixed into artificial soil containing 10 % peat. Artificial soil without test item was used as control. 40 test organisms with an individual weight between 0.40 - 0.54 g, divided into four replicates, were tested per test item concentration and control.

A statistically significant mortality was observed in the test item concentrations 100 and 316 mg/kg soil dry weight after 14 days of exposure, whereas no mortalities were observed in the control and the test item concentrations 3.16 to 31.6 mg/kg soil dry weight. Consequently, the NOEC was determined to be 31.6 mg/kg soil dry weight and LOEC was determined to be 100 mg/kg soil dry weight.

In terms of body weight loss, the NOEC was determined to be 31.6 mg/kg soil dry weight and LOEC was determined to be 100 mg/kg soil dry weight.

The LC50 for mortaility was 133 (70.1 - 262) mg/kg dw. The EC50 for body weight was 129 (88.3 - 185) mg/kg dw. As a worst-case these values observed for EDTA-FeNa will not be corrected for the MW difference.

One additional acute toxicity study with earthworms is available for EDTA-H4 (Edwards, 2009). The EC50 was determined at 156 mg/kg soil dry weight. This corresponds to 204.6 mg/kg soil dw EDTA-FeK.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

129 mg/kg soil dw

Additional information

The hazard assessment of EDTA-Fe(OH)K2 reveals neither a need to classify the substance as dangerous to the environment, nor is it a PBT or vPvB substance. The substance is expected to have a low potential for adsorption based on its ionic structure under environmental relevant pH conditions and its low log Kow , the low log Kow also indicates that the substance will not be bioaccumulative. There are no further indications that the substance may be hazardous to the environment.