Ethylenediamine-N,N'-di(acetic acid)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-24 to 2018-04-26

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item Ethylenediaminediacetic acid (ED2A-H2) was analytically verified via LC/MS-MS in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in the limit concentration and in the control.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Standard exposure duration

Hardness:

267 mg CaCO3/L

Test temperature:

18 - 22 °C, constant within ± 1 °C

pH:

8.06 control and 7.89 in 100 mg/L at start
7.71 control and 7.39 in 100 mg/L at end

Dissolved oxygen:

9.04 - 9.18 mg/L at start
8.19 - 8.24 mg/L at end

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal: 0 and 100 mg/L
The measured concentrations of the test item were within ± 20% of the nominal concentration. This indicates that the test item concentrations were successfully maintained for the duration of the test. Therefore, all effect values given are based on the nominal test item concentrations.

Details on test conditions:

Limit concentration A limit concentration of 100 mg/L of the test item was tested.
The limit concentration was selected based on the results of a non-GLP preliminary range finding test. For results, see Annex II.

Preparation of the The limit concentration (100 mg/L of the test item were weighed out)
limit concentration was freshly prepared with dilution water (Table 2) prior to the start of the exposure (0 hours). The test item solution was mixed thoroughly by manual agitation.

Control Dilution water without test item incubated under the same conditions as the test groups

Test method The study was performed under static conditions.

Test duration: 48 hours
Test vessels : Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
Test volume: 20 mL
Dilution water Same composition as the culture medium (see Table 2)
Number of daphnids 20 daphnids, divided into 4 replicates, each with 5 daphnids were
and replicates used for the limit concentration and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization Acclimatization was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by pipette.

Test temperature (target) 18 - 22 °C, constant within ± 1 °C

Illumination (target) Diffuse light, light intensity of max. 1500 lx

Photoperiod (target) 16/8 hours light/dark cycle

Feeding The daphnids were not fed during the study.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

99.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

99.2%

Basis for effect:

mobility

Results with reference substance (positive control):

Reference Test

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (see Table 2) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 7.2.

Reference item Potassium dichromate p.a. (SIGMA)
Purity 99.0%
Batch number MKBV0900V
Expiry date 2021-11-25
Test concentrations 1.00 - 2.00 - 4.00 mg/L
Ranges of validity
EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5),
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)

Test duration 2018-04-10 to 2018-04-11

Reported statistics and error estimates:

Methods of evaluation
The EC10 / 50 / 100-values after 24 and 48 hours for the test item were empirically derived from the observation data. Since no effects on Daphnia magna were observed in the limit concentration of the test item, no calculations were made. All effect concentrations (EC10 / 50 / 100) given were based on the nominal concentration of the test item.

EC-values and statistical analyses for reference item
An EC50-value was calculated for the reference item by sigmoidal dose-response regression from the best-fit values with the software GraphPad Prism. The respective 95 % confidence limits for the EC50 were empirically derived from the observation data as follows: The highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were used as 95% confidence limits, since calculation was not possible by the software.

Biological Data of the Definitive Test

The percentage of immobility, determined in the limit concentration and in the control after 24 and 48 hours, is given inTable3. The absolute numbers of immobile daphnids are presented in Table 4. Other adverse effects did not appear.

The study was performedunder static conditions.

 

Table3:      Immobilization Ratesafter 24 and 48 hours ofExposure in the Definitive Test

                (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

 

Table 4:      Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

                (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

Additional Observations during the Definitive Test

 

The limit concentration was colorless and visually clear throughout the exposure period.

Measured Exposure Concentrations during the Definitive Test

 

The concentration of the test item Ethylenediaminediacetic acid (ED2A-H2) was analytically verified via LC/MS-MS in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in the limit concentration and in the control. Details of the analytical method are presented in section 12.

The measured concentration of Ethylenediaminediacetic acid (ED2A-H2) in fresh medium was 91% of the nominal concentration at the start of the exposure (0 hours) and 108% of the nominal concentration at the end of the exposure (48 hours). The analytical results are presented in Table 5.

The measured concentrations of the test item were within ± 20% of the nominal concentration. This indicates that the test item concentrations were successfully maintained for the duration of the test.

 

Table 5:      Measured Concentrations of the Test Item Ethylenediaminediacetic acid (ED2A-H2) during the Definitive Test

Sampling		Fresh Media, 0 hours			Old Media, 48 hours
Nominal test item concentration [mg/L]		Ethylenediaminediacetic acid (ED2A-H2)
		Meas. conc. [mg/L]		%		Meas. conc. [mg/L]		%
100		91.4		91		108		108
Control		< LOQ			< LOQ

Meas. conc.= measured concentration of the test item, dilution factor taken into account

%                  =percentage of the nominal concentrationof the test item

LOQ             = limit of quantification of the analytical method (1.00 mg test item/L)

 

 

Test of the Reference Item

 

The percentage of immobility for the reference item potassium dichromate (Sigma-Aldrich, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2018‑04-10 to 2018-04-11. For results ofthe most recent of the monthly performed reference tests, see Table 6.

 

Table 6:      EC₅₀-Value (with Confidence Limits) of the Reference Item Potassium dichromate

                     based on the nominal concentrations [mg/L], (0 - 24 hours)

	Current Study	Valid Range
EC50	1.89 mg/L	0.6 - 2.4 mg/L, acc. toAQS P 9/2 (02/2000); clone 5 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
Confidence limits	1.00 - 4.00 mg/L

 

Validity criteria fulfilled:

yes

Remarks:

See overall remarks

Conclusions:

In the limit concentration of 99.2 mg a.i. /L of the test item Ethylenediaminediacetic acid (ED2A-H2), no effects on Daphnia magna were observed.
Based on the nominal concentrations of the test item Ethylenediaminediacetic acid (ED2A-H2), the 48 hours-EC10 / 50 / 100 for Daphnia magna was > 99.2 mg a.i./L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Ethylenediaminediacetic acid (ED2A-H2) (batch number:FC-C 12874) were determined at the test facility according to OECD 202 (2004) from 2018-04-24 to 2018-04-26.

The test item is an yellow powder that has a water solubility of approximately 10 g/L. A limit concentration of 100 mg/Lwas tested in a limit test under static conditions over a period of 48 hours. The limit concentration was colorless and visually clear throughout the exposure period.

Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the limit concentration and the control.

The concentration of the test item Ethylenediaminediacetic acid (ED2A-H2) was analytically verified via LC/MS-MS in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in the limit concentration and in the control. Details of the analytical method are presented in section 12.

The measured concentration of Ethylenediaminediacetic acid (ED2A-H2) in fresh medium was 91% of the nominal concentration at the start of the exposure (0 hours) and 108% of the nominal concentration at the end of the exposure (48 hours). The analytical results are presented inTable 5.

The measured concentrations of the test item were within ± 20% of the nominal concentration. This indicates that the test item concentrations were successfully maintained for the duration of the test. Therefore, all effect values given are based on the nominal test item concentrations. For results, please refer toTable below.

The validity criteria of the test guidelines were fulfilled.

 

Table:      EC₁₀-, EC₅₀- and EC₁₀₀-Values based on Nominal Concentration of the Test Item

Effect values	Test duration [hours]	Ethylenediaminediacetic acid (ED2A-H2) Nominal concentration of the test item, analytically verified [mg/L]
EC10/ EC50/ EC100	24	> 100
	48	> 100

 

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure according to German national standards (DIN)

Justification for type of information:

Study is read across and slightly updated and presented under "type of information" as experimental study until the lead company has updated the Source RSS
Read across justification is presented in IUCLID chapter 13

Qualifier:

according to guideline

Guideline:

other: DIN 38412, part 11

Principles of method if other than guideline:

STATISTICS
Spearman-Kaerber (1984)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The stock solution was prepared by dissolving 2000 mg test substance in dilution water. The test concentrations were then prepared by dilution with test water.

Test organisms (species):

Daphnia magna

Details on test organisms:

Strain: STRAUS
Breeder: Dr. U. Noack-Laboratorium
Hildesheim Source: Bundesgesundheitsamt, Berlin
Method of breeding: single breeding

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

21 °C

Dissolved oxygen:

8.0 - 8.2 mg/L

Nominal and measured concentrations:

Test concentrations (nominal): 58, 100, 180, 320 and 580 mg/L

Details on test conditions:

TEST SYSTEM
Type: open beaker (50 mL)
Aeration: none
No. of organisms per vessel: 5
No. of replicates: 4
Controls: negative control
Highest concentration after pH adjustment

TEST MEDIUM / WATER PARAMETERS
Dilution water: according to DIN 38412 L11, 8.2
Conductivity: 658 µS/cm
Ca/Mg ratio: 5:1
Na/K ratio: 20:1

TEST CONDITIONS
Adjustment of pH: none
Illumination: 10 uE/qm*s pH
During test: 3.85 - 7.90

The number of immobilized animals were recorded after 3, 6, 24, and 48 hours after test start.
The parameters pH, temperature and oxygen concentration were determined at the end of the test.

Reference substance (positive control):

yes

Remarks:

Potassium chromate

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

140 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 100-180 mg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No pH effect observed.

Results with reference substance (positive control):

Reference substance: Potassium chromate
EC50 (24 h): 0.9 mg/L
Date: 12 Sep - 13 Sep 1989

pH values at the end of the test
--------------------------------
580 mg/L             3.85
320 mg/L             4.02
180 mg/L             4.40
100 mg/L             7.30
 58 mg/L             7.73

control              7.90
control-pH           7.49
--------------------------------


Nominal       Mean number of mobile daphnids after
conc.               24h                   48h     
[mg/l]
-----------------------------------------------------------
 580                0/20                  0/20     
 320                0/20                  0/20    
 180                0/20                  0/20
 100               20/20                 19/20
  58               20/20                 19/20
 
 control           20/20                 19/20   
pH-control          0/20                  0/20
-----------------------------------------------------------

Description of key information

With an EC50>100 mg/L for daphnia, H2 -ED2A is acutely not harmful to aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

In the limit test at a concentration of 100 mg/L of Ethylenediaminediacetic acid (ED2A-H2), no effects on Daphnia magna were observed.

Based on the nominal concentrations of Ethylenediaminediacetic acid (ED2A-H2), the 48 hours-EC_{10 / 50 / 100} for Daphnia magna was >100 mg/L or >99.2 mg a.i./L.

The results observed ED2A-H2 are consistent with the other ecotoxicity endpoints. The available data from H4 -EDTA which were initally used for read-across to fill the datagap are now used in combination with the ED2A-H2 results for bridging purposes. All ecotox, fate and phys-chem data from H4 EDTA, Na2H2 EDTA, Na4 EDTA, HEDTA, H2 ED2A etc have been collated in a document which is compiled to justify the read across. This document is included in IUCLID 6, Chapter 13.

Tests on acute toxicity of H4 EDTA with Daphnia magna resulted in EC50 values well above 100 mg/L [EU Risk Assessment, 2004]. Exemplarily in an non-GLP study according to DIN 38412 the measured EC50-value for Na2H2 EDTA was 140 mg/L [BASF AG, 1989] which equals an EC50 of 120 mg/L for H4 EDTA