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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29-30 June 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study according to guideline and under GLP, some data on identity and purity, batch no. given but no CoA attached. Some phys-chem properties, no actual concentrations. When data on composition >=99.9%, reliability can be considered 2 (reliable with restrictions).
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
No actual concentrations measured
GLP compliance:
yes
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.H20, 0.2 g MgS04.7H20, 2.8 g K2HP04. 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 I M NaOH diluting to approximately 30 ml with deionized water, adding under stirring 0.5 M H2S04 to the point of incipient precipitation and finally diluting the mixture to 0.1 liter with deionized water (deviation of study plan; 100 ml prepared instead of 1 litre) . The stock solution of EDTA was prepared by adding 1.33 g Na2H2EDTA 2 H20 and 0.525 g CaCl2 2 H20 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 litre. 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: 125-250-500 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 um 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
Duration:
30 min
Dose descriptor:
EC50
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
Conclusions:
Restrictions as to composition of test material, no CoA. Study well performed, results are valid. EC50 (> 500 mg/L) can therefore be considered reliable.
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, 3,5dichlorophenol (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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer-reviewed data from EU Risk Assessment Report
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Equimolar amounts of CaCl2 were added, thus CaEDTA was formed in the stock solutions.
Test organisms (species):
activated sludge, domestic
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Nominal and measured concentrations:
Na2H2EDTA was used in concentrations of 125, 250, and 500 mg/L (referred as H4EDTA).
Details on test conditions:
Type: aquatic
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
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
At the highest test concentration (500 mg/L) a significant inhibition of the respiration rate could not be detected after 30 minutes. It can be concluded that both the EC10 and NOEC values are above 500 mg/L.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance.
Adequacy of study:
key study
Justification for type of information:
Refer to the Analogue Approach Justification document provided in Section 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
Reason / purpose for cross-reference:
read-across source
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
H4EDTA
Basis for effect:
inhibition of total respiration
Remarks on result:
other: RA-A, CAS 139-33-3, van Ginkel & Stroo, 2000, Respiration inhibition, 3 h, RL2
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: RA-A, CAS 139-33-3, van Ginkel & Stroo, 2004, Respiration inhibition, 30 min, RL2
Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
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
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.

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
Specific details on test material used for the study:
OTHER SPECIFICS
Purity/content: 40 % test substance, 55 % water, 5 % salts
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
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
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
Respiration inhibition at 1000 mg/L: 4.4 %

EC20 (relating to test compound) > 400 mg/L

Description of key information

In conclusion EDTA species are of low toxicity to (activated sludge) microorganisms and the degradation process in sewage treatment plants is not expected to be inhibited.

Key value for chemical safety assessment

Additional information

Since no studies investigating the toxicity trisodium hydrogen EDTA (CAS 150-38-9) to activated sludge microorganisms are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to structurally related EDTA species was conducted. This read-across is justified in detail in the overall summary (IUCLID Section 6.1) and within the analogue justification in IUCLID Section 13.

For the assessment of toxicity to microorganisms data from the source substances disodium dihydrogen EDTA (CAS 139-33-3), edetic acid (CAS 60-00-4) and tetrasodium EDTA (CAS 64-02-8) were used.

The first study (van Ginkel, 2000) with the source substance disodium dihydrogen EDTA the toxicity towards activated sludge microorganisms was determined in accordance with OECD 209 (GLP). The toxicity of the substance to activated sludge was determined by measuring the respiration rate after a contact time of 30 min at various concentrations of the test substance (125 -500 mg/L based on H4-EDTA). Equimolar amounts of CaCl2 were added, thus Ca-EDTA was formed in the stock solutions. From the results it is clear, that the substance does not significantly inhibit the respiration of activated sludge microorganisms up to a nominal concentration of 500 mg/L resulting in an EC50 (30 min) > 500 mg/L. The test is valid as shown by the EC50 of the reference compound, 3,5-dichlorophenol of 6.4 mg/L and control respiration rates which are within 15%.
This result is supported by an additional study with the same substance under comparable test conditions (van Ginkel, 2004). In this study the same EC50 (30 min) of > 500 mg/L resulted.

Both results are supported by a study performed according to ISO 8192 with the source substance tetrasodium EDTA. Activated sludge from an industrial sewage treatment plant did not cause inhibition of total respiration resulting in an EC10 (30 min) of > 1000 mg/L (nominal).

An additional study is available investigating the effects of edetic acid on nitrification by microorganisms (Hu et al, 2003). In the 24 h nitrification inhibition test the EC50 of H4-EDTA to microorganism was determined to be 2.4 mM/L, which is equivalent to 700 mg/L. The study did not follow a standard guideline but the result indicates a low toxicity to nitrifying microorganisms.

In conclusion EDTA species are of low toxicity to (activated sludge) microorganisms and the degradation process in sewage treatment plants is not expected to be inhibited.