Trisodium [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]manganate(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:

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 (DTPA-FeHNa) and the target substance (DTPA-MnNa3) both have the same chelating agent – DTPA, the main difference is metal cation – Fe+3 and Mn+2 respectively.
More explanation can be found in Section 13 assessment Reports – “RA Statement” and “Justification of read-across within the aminocarboxylic acid (ethylenediamine-based) chelants chemical category - Environmental fate and ecotoxicology”.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

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.