Disodium dihydrogen ethylenediaminetetraacetate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000-06-29 to 2000-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Origin: Acros organics, 's Hertogenbosch, The Netherlands
- Batch no. AO 10223201
- Appearance: white powder
-Purity: > 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Stability: stable at test conditions
Solubility in water: soluble at test concentrations
Storage: at room temperature in the dark

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The synthetic sewage feed was made by dissolving the following amounts of substances in one liter of deionized water: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCI, 0.4 g CaCI2.2.H2O, 0.2 g MgSO4.7H20, 2.8 g K2HPO4. The EDTA and 3.5-dichlorophenol were added to the incubation vessels using stock solutions of 1.0 and 0.5 g/L, respectively. The stock solution of 3.5-dichlorophenol was prepared by dissolving 0.05 g 3.5-dichlorophenol in 1 mL of 1 M NaOH diluting to approximately 30 mL with deionized water, adding under stirring 0.5 M H2SO4 to the point of incipient precipitation and finally diluting the mixture to 0.1 L with deionized water (deviation of study plan; 100 mL prepared instead of 1 L) . The stock solution of EDTA was prepared by adding 1.33 g Na2H2EDTA 2 H2O and 0.525 g CaCl2 2 H2O to approximately 900 mL of deionized water. The pH of this solution was titrated to 6.8 with a 1 N NaOH solution. Finally this mixture was diluted to 1 L. The concentration H4EDTA in this solution was 1 g/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Secondary activated sludge was obtained from the WVVTP Nieuwgraaf in Duiven (2000.06.29). The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. Prior to use the activated sludge was diluted with tap water (1.5x) and homogenized for two minutes in a lab blender 400. The dry weight of the activated sludge in the incubation vessels was 1.1 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Post exposure observation period:

None

Hardness:

No specific data

Test temperature:

21 °C

pH:

7.1 - 7.5

Dissolved oxygen:

Is parameter of interest in this study

Nominal and measured concentrations:

Nominal concentrations: 125, 250 and 500 mg/L expressed as H4EDTA

Details on test conditions:

The test was performed in 250 to 300 mL Erlenmeyers. The homogenized activated sludge was incubated in a shaking water bath (100 Hz, 20°C) for 30 minutes with various concentrations of the test compound and synthetic sewage.
The respiration rates of the activated sludge were measured in a biological Oxygen Monitor YSI 5300 (Yellow Springs Instruments, Ohio, USA). The BOM consisted of a thermostated vessel with a magnetic stirrer and an oxygen electrode. The electrode to measure the oxygen depletion tightly closed the vessel. The volume of the vessel was 10 mL and the temperature in the vessel was 20°C. The pH was measured using a Consort pH meter (Salm en Kipp BV, Breukelen, The Netherlands). The temperature was measured with a thermometer. The dry weight of the inoculum was determined by filtrating 100 mL of the activated sludge over a preweighed 12 µm Schleicher and Schull filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. Dry weight was calculated by subtracting the weighed filters.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Key result

Duration:

30 min

Dose descriptor:

EC10

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

H4EDTA

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Only minor (5%) inhibition of the respiration was observed in the medium dose level (250 mg/L).
In the 125 and 500 mg/L minor (5-8%) stimulation was observed.

Results with reference substance (positive control):

Inhibition was dose dependent, with an EC50 of 6.4 mg/L, within prescribed range (5-30 mg/L) for an exposure of 3 hours.

Reported statistics and error estimates:

The EC values were computed from the best fitted line (least square method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the substance. All computations were performed with the program TOXCALC(TM) on a personal computer.

Concentration (mg/L)	Activity mg O2/L min	% inhibition
Control	0.29	-
Control	0.28	-
125	0.31	0 (-9)
250	0.27	5
500	0.30	0 (-5)

Validity criteria fulfilled:

yes

Executive summary:

An activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice. The test is valid as shown by the EC50 of the reference compound, 3,5 dichlorophenol (6.4 mg/L) and control respiration rates which are within 15% of each other. The toxicity of EDTA to activated sludge was determined by measuring the respiration rate after a contact time of 30 minutes at various concentrations of the test substance (125 - 500 mg/L). The inhibitory effect of EDTA at a particular concentration was expressed as a percentage of the two controls. From the results it is clear, that EDTA does not significantly inhibit the respiration of activated sludge up to a nominal concentration of 500 mg/L. EC20, EC50, EC80 >500 mg/L.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-06-2010 - 15-06-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

The ethylenediaminetetraacetic acid, ferric-sodium complex, trihydrate stock solution was prepared by stirring 1.00 g ethylenediaminetetraacetic acid, ferric-sodium complex, trihydrate in 500 mL of deionized water. The ethylenediaminetetraacetic acid, ferric-sodium complex, trihydrate stock solution was a clear solution.
pH stock solution = 5.3

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Unadapted secondary activated sludge was obtained (14-06-2010) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The dry weight of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The dry weight of the activated sludge in the incubation vessels was 1.7 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.1 - 21.1°C.

pH:

6.9 - 7.2.

Dissolved oxygen:

aerobic during test

Nominal and measured concentrations:

Nominal: 40 mg/l, 80 mg/l, 160 mg/l, 320 mg/l and 640 mg/l

Details on test conditions:

-The test was performed in 250 to 300 ml Erlenmeyers. The homogenized activated sludge was incubated in a shaking water bath (100 rpm, 20°C) for 3 hours with various concentrations of the test compound and synthetic sewage. Synthetic sewage was added to the activated sludge (1.6 ml on a total volume of 50 ml): Synthetic sewage contained per liter: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4. Different concentrations (spaced by a factor two) of test compound and reference compound where added using stock solutions of respectively 2.0 g/l and 0.5 g/l. Tap-water was used to fill up the total test volume to 50 ml. The dry weight concentration of the activated sludge in the test was 1.7 g/l.
Controls without test substance (or reference compound) are measured at the beginning and at the end of the test. The inhibitory effect of the test substance/reference compound is expressed as a percentage of the mean of the two control respiration rates.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol. The tested concentrations of the reference compound 3,5-dichlorophenol where: 1.5, 3.0 mg/l, 5.9 mg/l,11.8 mg/l, 23.6 mg/l and 47.2 mg/l.

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 640 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 640 mg/l was the highest concentration tested

Details on results:

Validity of the test
The validity of the test is demonstrated by two criteria. First, the control respiration rates were within 15% of each other and secondly, the EC50 of the reference compound was 7 mg/L which is within the prescribed range of 5 to 30 mg/L .

Activated sludge respiration inhibition test
The inhibitory effect of ethylenediaminetetraacetic acid, ferric-sodium complex, trihydrate at a particular concentration is expressed as a percentage of the two controls. No inhibition of the respiration of the activated sludge was measured at the highest concentration tested, i.e. 640 mg/L.
Ethylenediaminetetraacetic acid, ferric-sodium complex, trihydrate is therefore considered not harmful to activated sludge.

Results with reference substance (positive control):

The EC50 of the reference compound was 7 mg/L at a contact time of 3 hours, respectively. These EC50 values is within the prescribed range of 5 to 30 mg/L.

Validity criteria fulfilled:

yes

Remarks:

The validity of the test is demonstrated by two criteria. First, the control respiration rates were within 15% of each other and secondly, the EC50 of the reference compound was 7 mg/L which is within the prescribed range of 5 to 30 mg/L .

Conclusions:

Test performed under GLP according guidelines. The test is valid as shown by the EC50of the reference compound, 3,5-dichlorophenol (7 mg/L
contact time 3 hours ) and the control respiration rates which are within 15% of each other.
No inhibition of the respiration of the activated sludge was measured at the highest concentration tested, i.e. 640 mg/L. Ethylenediaminetetraacetic
acid, ferric-sodium complex, trihydrate is therefore considered not harmful to activated sludge.

Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice.

The test is valid as shown by the EC₅₀of the reference compound, 3,5-dichlorophenol (7 mg/L) and the control respiration rates which are within 15% of each other.

The toxicity of ethylenediaminetetraacetic acid, ferric-sodium complex, trihydrate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of ethylenediaminetetraacetic acid, ferric-sodium complex, trihydrate at a particular concentration is expressed as a percentage of the two controls. No inhibition of the respiration of the activated sludge was measured at the highest concentration tested, i.e. 640 mg/L. Ethylenediaminetetraacetic acid, ferric-sodium complex, trihydrate is therefore considered not harmful to activated sludge

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure according to international standards (ISO)

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

activated sludge, industrial

Details on inoculum:

The activated sludge inoculum used in this study originated from an industrial wastewater treatment plant (BASF AG). The concentration of dry substance was 1 g/L.

Test type:

static

Water media type:

freshwater

Details on test conditions:

Type: aquatic

Key result

Duration:

30 min

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

30 min

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Respiration inhibition at 1000 mg/L: 4.4 %

Reference 4

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

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

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 650 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: EDTA, EDTA-FeNa, -Na4

Duration:

30 min

Dose descriptor:

other: EC20

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Reference 5

Endpoint:

activated sludge nitrification inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable, well documented study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Nitrification inhibition was evaluated in 24 h batch extant respirometric assays employing biomass from a continuously operated nitrifying enrichment reactor.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: EDTA (electrophoresis grade, Fisher Scientific, Fairlane, NJ, USA)

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

aerobic microorganisms

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

24 h

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2.4 mmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: nitrification inhibition

Validity criteria fulfilled:

not specified

Conclusions:

The concentration causing 50% inhibition was estimated at 2.4 mM/L for EDTA. Inhibition by EDTA reached its ultimate value within 6 h.

Executive summary:

In a 24 h nitrification inhibition test the EC50 of EDTA to microorganism from a continuously operated nitrifying enrichment reactor was determined to be 2.4 mM/L.

Description of key information

EC10 (0.5-3h) > 500 mg/L for respiration inhibition of activated sludge (OECD 209, WoE, read across)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

500 mg/L

Additional information

One study is available on the microorganism toxicity of Disodium dihydrogen ethylenediaminetetraacetate (CAS No. 139-33-3). The study was performed according to OECD guideline 209 and GLP using non-adapted activated sludge obtained from a municipal STP as inoculum (Akzo 2000). Activated sludge was exposed to the test substance in a static system for 30 minutes, at nominal concentrations between 125 and 500 mg/L (expressed as H4EDTA). Only minor (5%) inhibition of the respiration was observed in the medium dose level (250 mg/L). In the 125 and 500 mg/L minor (5-8%) stimulation was observed. Thus, the 30 min-EC10 is determined to be > 500 mg/L. In addition, studies with subcategory 1a member Tetrasodium ethylenediaminetetraacetate (CAS No. 64-02-8) and subcategory 1b member Sodium feredetate (CAS No. 15708-41-5) are considered as part of a Weight-of-evidence 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 study with Tetrasodium ethylenediaminetetraacetate (CAS No. 64-02-8) was performed according to ISO guideline 8192 using activated sludge from an industrial STP as inoculum (BASF 1988). Activated sludge was exposed to the test substance in a static system for 30 minutes, at a nominal concentrations of 1000 mg/L. Respiration inhibition of 4.4% was observed in the treatment; thus, the EC10 is determined to be > 1000 mg/L. The study with Sodium feredetate (CAS No. 15708-41-5) was also performed according to OECD guideline 209 and GLP using non-adapted activated sludge obtained from a municipal STP as inoculum (Akzo 2010). The inoculum was exposed to nominal test concentrations between 40 and 640 mg/L for 3h. No inhibition of the respiration of the activated sludge was measured up to the highest concentration tested, i.e. 640 mg/L. Thuy, the 3h-NOEC/EC10 is determined to be > 640 mg/L. Thus, based on the above-mentioned results, it can be concluded that Disodium dihydrogen ethylenediaminetetraacetate and the other subcategory 1 member are not harmful to aquatic microorganisms and an inhibition of its degradation activity in STPs is not anticipated. This low toxicity pattern is supported by Hu (2003), who investigated nitrification inhibition of edetic acid exposed over 24h to microorganisms. A 24h-EC50 was determined to be 2.4 mM/L, which is equivalent to 700 mg/L.

In absence of adverse effects to microorganism respiration observed in the available studies but aiming to apply a conservative environmental risk assessment, an EC10 value of 500 mg/L was used to derive a PNEC STP for Disodium dihydrogen ethylenediaminetetraacetate and all other Subcategory 1 members.