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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
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.

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

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 %
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
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
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
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

Since no studies are available on the microorganism toxicity of edetic acid (CAS No. 60-00-4), the assessment was based on studies conducted with other Subcategory 1a members Tetrasodium ethylenediaminetetraacetate (CAS No. 64-02-8) and Disodium dihydrogen ethylenediaminetetraacetate (CAS No. 139-33-3) as well as the Subcategory 1b member Sodium feredetate (CAS No. 15708-41-5) 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 Disodium dihydrogen ethylenediaminetetraacetate (CAS No. 139-33-3) 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. 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 edetic acid 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, a EC10 value of 500 mg/L was used to derive a PNEC STP for edetic acid and all other Subcategory 1 members.