Potassium; 2-[bis[2-[bis(carboxylatomethyl)amino]ethyl]amino]acetate; iron(3+)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

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

Adequacy of study:

key study

Study period:

03-12-2012 - 05-12-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed under GLP according the guideline meeting all validity criteria

Justification for type of information:

A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent (DTPA) as in a target substance (DTPA-FeK). The only difference between the target and the source substance is presence of potassium (K) cation instead Na+ cations. As potassium and sodium are the essential macro elements required by all forms of life, is considered not to influence the toxicological activity.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

toxicity to microorganisms

Type of information:

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

Adequacy of study:

supporting study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted following ISO guideline and sufficient data is available for interpretation of results.

Justification for type of information:

The underlying hypothesis for the read-across is that chelates have the same mode of action based on their ability to chelate, remove or add metal cations to body causing perturbation of body’s micronutrients balance.
The source substance is a chelating agent in a target substance. The only difference between the target and the main source substance is presence of iron (Fe) and potassium (K) cations instead H+ cations. As iron and potassium are an essential macro- and microelements required by all forms of life, is considered not to influence the toxicological activity.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD 302B Zahn-Wellens biodegradation test

Principles of method if other than guideline:

In this OECD 302B Zahn-Wellens biodegradation study, the toxicity of the test compound to the activated sludge microorganisms was assessed.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

Samples filtered through 0.45 micron pore-size filters.

Vehicle:

not specified

Details on test solutions:

Biological testing medium containing calcium, magnesium, and iron cations at 0.25, 0.1, and 0.001 mmoles/L.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The inoculum was either non-adapted activated sludge collected at the municipal water treatment plant in Prague or an activated sludge adapted at
different sludge age in the range of 5–30 days, which corresponds to the relations in medium- to low-load biological water treatment plants.

Test type:

other: Ready biodegradation study

Water media type:

not specified

Limit test:

no

Total exposure duration:

30 d

Post exposure observation period:

Not applicable.

Hardness:

No data

Test temperature:

No data

pH:

No data

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

100 mg/L DTPA (acid form)

Details on test conditions:

Two methods were combined for evaluating the complete biodegradability: the method of assaying dissolved organic carbon (EN ISO 7827) suitable for assessing ready biodegradability (Xo=30mg/L, So= DOC 50mg/L) and the Zahn-Wellens Test (EN ISO 9888) for assessing potential biodegradability (Xo=200mg/L, So= DOC 50mg/L). Xo is the concentration of activated sludge and So is the initial test material concentration as DOC. Ready biodegradability means degradation of compounds by nonadapted activated sludge or adapted
sludge at sludge age mean biomass retention time (MBRT) 5 days (lag-phase approximately till 10 days). Sludge age 5 days usually corresponds to conditions at most treatment plants. Potential biodegradability means, that tested compounds are degradable under certain conditions (e.g. sludge age longer than 5 days). The substances under study were the only sources of organic carbon for the microbes of the inoculum. The inoculum was either non-adapted activated sludge collected at the municipal water treatment plant in Prague or an activated sludge adapted at different sludge age in the range of 5–30 days, which corresponds to the conditions in medium- to low-load biological water treatment plants

Reference substance (positive control):

not specified

Duration:

30 d

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: DOC Removal

Details on results:

Checks for inhibition and for abiotic processes were performed in parallel with biodegradability tests according to standards (EN ISO 7827 and EN ISO 9888). The results of these checks were negative in all cases. At the concentrations under study, the substances had no inhibitory effects on biological processes and no evidence of abiotic decomposition was obtained.

Results with reference substance (positive control):

Not available

Reported statistics and error estimates:

Not available

Validity criteria fulfilled:

not specified

Conclusions:

DTPA did not biodegrade in municipal activated sludge with sludge ages ranging from 5 to 30 days. DTPA was not inhibitory to biological processes at a concentration of 100 mg/L (acid form).

Executive summary:

DTPA did not biodegrade in municipal activated sludge with sludge ages ranging from 5 to 30 days. DTPA was not inhibitory to biological processes at a concentration of 100 mg/L (acid form).

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

toxicity to microorganisms

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:

other: Non GLP study with adequate documentation.

Justification for type of information:

The underlying hypothesis for the read-across is that chelates have the same mode of action based on their ability to chelate, remove or add metal cations to body causing perturbation of body’s micronutrients balance.
The source substance is a chelating agent in a target substance. The only difference between the target and the main source substance is presence of iron (Fe) and potassium (K) cations instead H+ cations. As iron and potassium are an essential macro- and microelements required by all forms of life, is considered not to influence the toxicological activity.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Batch and continuous aerobic biodegradation tests using high concentrations of DTPA and EDTA.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Samples were drawn using a sterile syringe and filtered through a 0.45 Jim disposable filter. These samples were diluted with organic free water to a sample concentration of approximately 20 mg/L of total organic carbon.

Vehicle:

no

Details on test solutions:

Biological degradation experiments were conducted wtih DTPA using batch tests. The batch experiments were conducted as follows. The 250 ml Erlenmeyer flasks were filled with 125 ml of substrate amended with a nutrient medium spiked with 5 ml of inoculum for a total volume of 130 ml, leaving 120 ml of head space in each flask. These flasks were incubated at 20°C on a continuous shaker table. Samples were drawn using a sterile syringe and filtered through a 0.45 Jim disposable filter. These samples were diluted with organic free water to a sample concentration of approximately 20 mg/L of total organic carbon.

Test organisms (species):

activated sludge

Details on inoculum:

Biological inocula were obtained from 4 sources. The first source was a pulp mill aerated lagoon that had en treating effluent containing DTPA for more than 5 years. The second source was from a 'craft pulp mill activated sludge system. The third inoculum came from an activated sludge process at a municipal water treatment plant. The fourth inoculum came from soil samples taken from a rose garden. Inocula were restored at 5°C until used and were stored no longer than 24 hours prior to inoculation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

20°C

pH:

6.9 to 7.4

Dissolved oxygen:

5 and 9 mg/L

Nominal and measured concentrations:

Measured concentrations of DTPA acid were approximately 400 and 725 mg/L in separate biodegradation tests based on TOC analyses.

Details on test conditions:

Reaction mixtures were incubated at 20°C on a continuous shaker table.

Reference substance (positive control):

yes

Details on results:

DTPA did not degrade in the batch experiments with the 4 different inocula. Pre-exposure of the pulp mill inocula to triethanolamine and nitrilotriacetic acid also did not promote degradation of DTPA. Authors did not report microbial inhibition as cause for lack of biodegradation since triethanolamine was rapidly degraded upon repeated additions to the reaction mixtures. Thus DTPA acid was not inhibitory at a concentration of 400 mg/L in reaction mixtures prepared with inocula from pulp mill activated sludge, municipal activated sludge, and soil. DTPA acid was not inhibitory at a concentration of 725 mg/L in reaction mixtures prepared with inocula from pulp mill activated sludge with prior exposure to DTPA.

DTPA did not degrade in the batch experiments with the 4 different inocula. Pre-exposure of the pulp mill inocula to triethanolamine and nitrilotriacetic acid also did not promote degradation of DTPA. Authors did not report microbial inhibition as cause for lack of biodegradation since triethanolamine was rapidly degraded upon repeated additions to the reaction mixtures. Thus DTPA acid was not inhibitory at a concentration of 400 mg/L in reaction mixtures prepared with inocula from pulp mill activated sludge, municipal activated sludge, and soil. DTPA acid was not inhibitory at a concentration of 725 mg/L in reaction mixtures prepared with inocula from pulp mill activated sludge with prior exposure to DTPA.

Validity criteria fulfilled:

not applicable

Conclusions:

DTPA acid was not inhibitory at a concentration of 400 mg/L in reaction mixtures prepared with inocula from pulp mill activated sludge, municipal activated sludge, and soil. DTPA acid was not inhibitory at a concentration of 725 mg/L in reaction mixtures prepared with inocula from pulp mill activated sludge with prior exposure to DTPA.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

minor deviation

Principles of method if other than guideline:

Minor deviation: The average oxygen uptake rate for the control replicates was slightly lower than prescribed in the guideline

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

EC Number:

235-627-0

EC Name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

Cas Number:

12389-75-2

Molecular formula:

C14-H18-Fe-N3-O10.H.Na

IUPAC Name:

Iron(3+) ion sodium 5-[bis(carboxylatomethyl)amino]-3-{[bis(carboxylatomethyl)amino]methoxy}pentanoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

A sample of diethylenetriaminepentaacetic acid, ferric sodium complex (see annex 2) was received from Akzo Nobel Functional Chemicals BV on 08-11-2012. The following test substance data were submitted by the sponsor, who accepted full responsibility for the validity thereof.
Name Diethylenetriaminepentaacetic acid, ferric sodium complex
Product name Dissolvine DFe-11 (Rexolin D12)
Abbreviation DTPA-FeHNa
CAS No. 12389-75-2
Purity See certificate of analysis
Batch/lot No. CFC 10333 (429H0352)
Appearance Yellow solid
Solubility in water soluble in water
Volatile No
Stability stable under test conditions
Storage In well closed container at ambient temperature in the dark
The concentrations cited in this report refer to the as-received sample of diethylenetriaminepentaacetic acid, ferric sodium complex.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Unadapted secondary activated sludge was obtained (04-12-2012) 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 suspended solids (dry weight) content of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The activated sludge was aerated and fed overnight with OECD synthetic sewage concentrate at 50 mL/L prior to use on the day following collection. The suspended solids concentration of the activated sludge in the incubation vessels was 1.5 g dry weight/L.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

Temperature of the reaction mixtures after the incubation period ranged from 20.0 to 20.7°C.

pH:

The pH of the reaction mixtures after the incubation period ranged from 6.6 to 7.0.

Dissolved oxygen:

aerobic during test

Nominal and measured concentrations:

The nominal concentrations of 3,5-Dichlorophenol tested were: 2.5, 5.0, 10.0, 20.0 and 40.0 mg/L
The nominal concentrations of diethylenetriaminepentaacetic acid, ferric sodium complex were: 40, 80, 160, 320, 640 and 1280 mg/L.

Details on test conditions:

Test conditions:
The test was performed in 0.30 liter Erlenmeyer flasks with a total working volume of 50 ml. 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 feed and stocks:
The synthetic sewage feed was made by dissolving the following amounts of substances in 1 liter of deionized water:
16 g peptone,
11 g meat extract,
3 g urea,
0.7 g NaCl,
0.3 g CaCl2,
0.2 g MgSO4.7H2O,
2.8 g K2HPO4.
In the incubation vessels 1.6 mL of the synthetic sewage feed is added in a total volume of 50 mL.

Diethylenetriaminepentaacetic acid, ferric sodium complex and 3,5-dichlorophenol were added to the incubation vessels using stock solutions of approximately 2.0 and 1.0 g/L respectively. The stock solution of diethylenetriaminepentaacetic acid, ferric sodium complex was prepared by dissolving 2.00 g in 1000 mL deionized water. The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1031 g of 3,5-dichlorophenol in approximately 40 mL of deionized water with the addition of a few drops of 3 M NaOH. Finally the pH was corrected with H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 280 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Test conditions
The pH of the reaction mixtures after the incubation period ranged from 6.6 to 7.0. Temperature varied from 20.0 to 20.7°C. These conditions allow respiration of the activated sludge used.

Validity of the test
The validity of the test is demonstrated by three criteria. First, the prescribed oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements, was not met. The inoculum used was obtained from a treatment plant which is operated at a low organic sludge load and as a consequence has a low oxygen uptake rate. Operating at low organic sludge loads is nowadays very common. Nonetheless the average oxygen uptake rate for the control replicates was 20 mg O2/g dry weight/hour, which is slightly lower than the prescribed rate. This does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The slightly lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test. Second, the coefficient of variation of the replicates of the control oxygen uptake rates was 3%, which shows a high precision and fulfills the maximum prescribed variation of <30%. Third, the EC50 of the reference compound for activated sludge after 3 hours contact time is 9 mg/L, which is within the prescribed range of 2 to 25 mg/L (Table I and Figure 1).

Activated sludge respiration inhibition test
The inhibitory effect of diethylenetriaminepentaacetic acid, ferric sodium complex at a particular nominal concentration is expressed as a percentage of the two controls. From these results EC values were calculated. The calculated respiration rates and inhibition percentages of diethylenetriaminepentaacetic acid, ferric sodium complex are presented in Table II and the results of the graphical probit analysis are shown in Figure 2. Computation of the EC confidence limits, on the basis of Fieller's theorem (Zerbe, 1987), was not possible for the obtained dose response curve. The EC50 of diethylenetriaminepentaacetic acid, ferric sodium complex for activated sludge after 3 hours contact time is >1280 mg/L. Diethylenetriaminepentaacetic acid, ferric sodium complex is therefore considered not harmful to activated sludge.

Results with reference substance (positive control):

The EC50 of the reference compound (3,5-dichlorophenol) was 9 mg/L which is within the prescribed range of 2 to 25 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see details on results

Conclusions:

Test performed under GLP, according guideline meeting validity criteria.
The test is valid as shown by the EC50 of the reference compound, 3,5-dichlorophenol (9 mg/L) and the coefficient of variation of the replicates of the
control oxygen uptake rates (3%). The average oxygen uptake rate for the control replicates is slightly lower (20 mg O2/g dry weight/hour) than the
prescribed rate (> 20 mg O2/g dry weight/hour). This does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The slightly lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test.
The toxicity of diethylenetriaminepentaacetic acid, ferric sodium complex to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of diethylenetriaminepentaacetic acid, ferric sodium complex at a particular nominal concentration is expressed as a percentage of the two controls. The EC50 of diethylenetriaminepentaacetic acid, ferric sodium complex for activated sludge after 3 hours contact time is >1280 mg/L. Diethylenetriaminepentaacetic acid, ferric sodium complex 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 substance, 3,5-dichlorophenol (9 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (3%). The average oxygen uptake rate for the control replicates is slightly lower (20 mg O₂/g dry weight/hour) than the prescribed rate (>20 mg O₂/g dry weight /hour). This does not influence the sensitivity of the activated sludge as shown by comparable EC₅₀values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The slightly lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test. The toxicity of diethylenetriaminepentaacetic acid, ferric sodium complex to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of diethylenetriaminepentaacetic acid, ferric sodium complex at a particular nominal concentration is expressed as a percentage of the two controls. The EC₅₀of diethylenetriaminepentaacetic acid, ferric sodium complex for activated sludge after 3 hours contact time is >1280 mg/L. Diethylenetriaminepentaacetic acid, ferric sodium complex is therefore considered not harmful to activated sludge.