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Toxicity to microorganisms

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Endpoint:
toxicity to microorganisms
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
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
Key result
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 H4EDTA. Inhibition by EDTA reached its ultimate value within 6 h.
Executive summary:

In a 24 h nitrification inhibition test the EC50 of H4EDTA 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:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
29-30 June 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reasonably documented study conducted in accordance with recognized test methods
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
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
Key result
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:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
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
Key result
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
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
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 based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
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
Key result
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
> 400 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
at 40%
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 %

EC20 (relating to test compound) > 400 mg/L
Conclusions:
Observed respiration inhibition at a nominal concentration of 1000 mg/L (400 mg/L relating to substance at 40% AI) was 4.4%
Executive summary:
Observed respiration inhibition at a nominal concentration of 1000 mg/L (400 mg/L relating to substance at 40% AI) was 4.4%. The IC10 can be regarded as being greater than 400 mg/L.

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
589 mg/L

Additional information

There are no reliable studies available on the toxicity of trisodium HEDTA to aquatic microorganisms, however the effects of disodium EDTA and EDTA were investigated in activated sludge. Overall, low toxicity was observed. In the key study, a 30 min EC50 >500 mg/L was found, expressed as H4-EDTA. Recalculated as Na3-HEDTA, this is equivalent to ca. 589 mg/L (based on a molecular weight of 292.24 for EDTA and 344.20 for Na3-HEDTA). This value was retained for the CSA.