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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 - 14 June 2007
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
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Details on sampling:
After the test, the contents of the test vessels were poured into flat bottom flasks. Then oxygen measurements were conducted.
Vehicle:
no
Details on test solutions:
The dilution medium (deionised water) will be adapted to test temperature. For each test concentration, the dilution medium will be combined with the synthetic sewage feed (16 mL) in a way that a total volume of 300 ml will be obtained. The test item is weighed into a vessel and transferred to the test medium. The volume of the test solution will be large enough to prepare each test concentration level. Then 200 ml of the microbial inoculum were added. The total mixture for each concentration and the two controls was 500 mL.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge from a sewage treatment plant will be used as the microbial inoculum for the test. The activated sludge is obtained preferentially from a sewage work treating predominantly domestic sewage.
On the day the activated sludge is obtained from the sewage treatment plant it was washed with reconstituted water. After centrifugation of the sludge the supernatant was decanted. This procedure was repeated three times.
The final concentration of active sludge in the test medium was 1.6 g/l.
If the activated sludge was not used the day of the collection, 50 ml synthetic sewage feed was added to each litre of the activated sludge and it was aerated with clean, oil-free air and kept at a temperature of 20 ± 2°C. At the end of every storage day the sludge will be fed with 50 ml/l of synthetic sewage feed (maximum storage = 4 days).
The used microbial inoculum had a mixed liquor suspended solids level of 3.6 g/l. The final level in the test solustions was 1.44 g/l.

RECONSTITUTED WATER
According to OECD Guideline No. 203, prepared according the ECT-Standard Operation Procedure (SOP) A 2.1.
Used to keep the microbial inoculum before the period of the test.
Concentration of salts: 294.0 mg/l CaCl2.2H2O, 123.0 mg/l MgSO4.7H2O, 64.8 mg/l NaHCO3 and 5.75 mg/l KCl.
Water was prepared not longer than 4 weeks before it was used. During storage water was aerated.

SYNTHETIC SEWAGE FEED
Contains peptone, meat extract, urea, NaCl, CaCl2.2H2O, MgSO4.7H2O and K2HPO4.
Prepared not longer than 1 week before it was used.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
235.8 mg/l CaCO3
Test temperature:
19.1-19.7 °C
pH:
7.4-12.6 (before contact time)
7.8-10.6 (after contact time)
Dissolved oxygen:
8.7 mg/l (at the beginning of the test)
Salinity:
not applicable
Nominal and measured concentrations:
Nominal: 0, 62.5, 125, 250, 500 and 1000 mg/l
Details on test conditions:
Before use the pH of the activated sludge will be checked and adjusted if necessary to a pH 6.0-8.0 using sodium hydrogen carbonate (NaHCO3) solution.
Every 15 minutes one test vessel containing the test item will be prepared (starting with control 1 and ending with control 2):
- 16 ml of synthetic sewage feed was added to each test vessel.
- deionised water and/or the prepared stock solution was added to the test vessels to a total volume of 300 ml.
- the amount of test item necessary to result in the desired test concentration was added to the test medium and stirred.
- the pH was measured in the test medium.
- 200 ml microbial inoculum was added to each test vessel.
Test vessels: 1000 ml glass beakers.
Number of replicates per test item concentration: 1
Number of replicates in the control: 2
After 3 hours of incubation, the pH was measured. Afterwards the content of the vessels was poured into the measuring apparatus and the respiration rate was determined.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
229.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 192.4 mg/l. Upper 95%: 252.6 mg/l.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
300.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 273.4 mg/l. Upper 95%: 348.9 mg/l.
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
393.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 341.6 mg/l. Upper 95%: 548.1 mg/l.
Details on results:
The biological findings were closely related to the initial pH of the test solutions, which increased from 7.4 in the controls to pH 9.2, 10.0, 11.6, 12.2, 12.6 at 62.5, 125, 250, 500 and 1000 mg/L, respectively. Within the contact time of 3 hours, the initial pH was considerably lowered which is likely attributable to the reaction of the test item with CO2 in the medium to CaCO3. Therefore the initial pH is considered to be the main reason for the effects of the test item on the test organisms.
Results with reference substance (positive control):
The 3h- EC50 was in the accepted range of 5-30 mg/L : namely 7.8 mg/L.
Validity criteria fulfilled:
yes
Remarks:
The two control respiration rates within 15 per cent of each other: 0,4%. The EC50 (3 hours) of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/l: 7,8 mg/l.
Conclusions:
The biological findings (inhibition of respiration) were closely related to the initial pH of the test solutions.
Within the contact time of 3 hours, the initial pH was considerably lowered which is likely attributable to the reaction of the test item with CO2 in the medium to Calcium carbonate. Therefore the initial pH is considered to be the main reason for the effects of the test item in the test organisms.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 December 2009 - 11 December 2009
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
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was dispersed directly in water. Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately dispersed in approximately 250 mL of water and subjected to ultrasonication for approximately 15 minutes followed by prolonged mixing for 24 hours in order to maximise the dissolved test material concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L.
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.5 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.7 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not applicable
Hardness:
Approximately 140 mg/L as CaCO3
Test temperature:
21±1 °C
pH:
See Table 2
Dissolved oxygen:
See Table 1
Salinity:
No data
Nominal and measured concentrations:
Nominal test concentrations: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 500 mL conical flask
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener). After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 0.810 - 1.380 mg/L (average 1.041 mg/L)
- Particulate matter: No data
- Metals: Various different metals were present in the water and were all below the PCV (prescribed concentration or value for mandatory parameters and specified limit or value for non-mandatory parameters)
- Pesticides: 0.0 - 0.001 µg/L
- Chlorine (total): 0.080 - 0.610 mg/L (average 0.297 mg/L)
- Alkalinity: pH 7.26 - 8.24 (average 7.719)
- Ca/mg ratio: No data
- Conductivity: 251.0 - 542.0 µS/cm at 20 °C (average 421.385 µS/cm)


OTHER TEST CONDITIONS
- Light intensity: The test was conducted under normal laboratory lighting.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/L and 2.5 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.
Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
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:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 5%.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Observations made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10 and 32 mg/L contained slightly cloudy dispersions with particles of test material settled at the bottom of the vessel. The test concentrations of 100, 320 and 1000 mg/L contained cloudy dispersions with particles of test material settled on the bottom of the vessel.
Observations made after 30 minutes and 3 hours contact time for the test concentrations of 10 and 32 mg/L showed the vessels contained a dark brown dispersion with no undissolved test material visible. The test concentration of 100 mg/L contained a dark brown dispersion with test material visible at the bottom of the vessel. The test concentrations of 320 and 1000 mg/L contained a light brown dispersion with test material visible dispersed throughout.
Results with reference substance (positive control):
The 3 h EC50 for 3,5-dichlorophenol was 7.0 mg/L (95% confidence limits 5.3 - 9.3 mg/L).

Table 1: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal

Concentration

(mg/L)

Initial O2

(mg O2/L)

Measurement Period

(minutes)

Final O2Reading

(mg O2/L)

O2Consumption Rates

(mg O2/L/min)

% Inhibition

Control

R1

5.2

5

2.5

0.54

-

R2

5.2

5

2.2

0.60

-

Test Material

10

5.1

4

2.8

0.58

[2]

 -

32

5.2

5

2.4

0.56

2

 -

100

5.2

5

2.3

0.58

[2]

 -

320

5.5

5

2.6

0.58

[2]

 -

1000

5.9

6

2.5

0.57

0

3,5-dichlorophenol

3.2

6.9

10

3.0

0.39

32

10

7.2

10

4.8

0.24

58

 -

32

8.1

10

7.3

0.08

86

[Increase in respiration rate as compared to controls]

R1– R2= Replicates 1 to 2

Table 2: pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal

Concentration

(mg/l)

pH

0 Hours

3 Hours

Control

R1

7.4

7.8

-

R2

7.6

7.7

Test Material

10

7.6

7.6

-

32

7.6

7.6

-

100

7.6

7.7

-

320

7.6

7.7

-

1000

7.7

7.7

3,5-dichlorophenol

3.2

7.5

8.0

-

10

7.5

8.0

-

32

7.5

8.1

R1– R2= Replicates 1 to 2

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Common functional groups/mechanism of action.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target: Lime (chemical), hydraulic [CAS 85117-09-5; See section 1.2 for information on purity.
Source: calcium dihydroxide [CAS 1305-62-0; EC 215-137-3] 98.2%

3. ANALOGUE APPROACH JUSTIFICATION
In the environment, lime substances rapidly dissociate or react with water. These reactions, together with the equivalent amount of hydroxyl ions set free when considering 100mg of the lime compound (hypothetic example), are illustrated below:
Ca(OH)2 <-> Ca2+ + 2OH-
100 mg Ca(OH)2 or 1.35 mmol sets free 2.70 mmol OH-
Ca(OH)2 + Ca2SiO4 + CaCO3 + 3 H2O <-> 4Ca2+ + SiO2 + CO2 + 8OH-
100 mg lime (chemical) hydraulic or 0.27 mmol sets free 2.16 mmol OH-
It has to be noted that CO32- is not expected to directly release two hydroxyl ions under most environmental conditions (depends on CO2 concentrations and pH) and this is therefore a worst case assumption.
From these reactions it is clear that the effect of hydraulic lime (chemical) will be caused either by calcium or hydroxyl ions. Since calcium is abundantly present in the environment and since the effect concentrations are within the same order of magnitude of its natural concentration, it can be assumed that the adverse effects are mainly caused by the pH increase caused by the hydroxyl ions. Furthermore, the above mentioned calculations show that the base equivalents are within a factor 2 for hydraulic lime (chemical) and calcium hydroxide. As such, it can be reasonably expected that the effect on pH of lime (chemical) hydraulic is comparable to calcium hydroxide for a same application on a weight basis. Consequently, read-across from calcium hydroxide to lime (chemical) hydraulic is justified.

4. DATA MATRIX
Source: No studies available
Target: nominal EC50(3h) = 300.4 mg Ca(OH)2/L (OECD 209)
Reason / purpose for cross-reference:
read-across source
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
229.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 192.4 mg/l. Upper 95%: 252.6 mg/l.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
300.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 273.4 mg/l. Upper 95%: 348.9 mg/l.
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
393.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Lower 95%: 341.6 mg/l. Upper 95%: 548.1 mg/l.

Description of key information

Klimisch 1 study (Egeler et al. (2007): activated sludge respiration inhibition test for Ca(OH)2 according to OECD 209; nominal EC50(3h) = 300.4 mg Ca(OH)2/L .

Klimisch 1 study; activated sludge respiration inhibition test according to OECD 209; nominal EC50(3h) >1000 mg CaCO3/L (Youngs, 2010)

Key value for chemical safety assessment

EC50 for microorganisms:
300.4 mg/L

Additional information

The toxicity to aquatic microorganisms of calcium dihydroxide was assessed in a study performed to OECD TG 209 under GLP (Egeler et al. 2007). Activated sewage sludge was exposed to calcium dihydroxide at nominal test concentrations of 0, 62.5, 125, 250, 500 and 1000 mg/L for 3 hours. The biological findings were closely related to the initial pH of the test solutions, which increased from 7.4 in the controls to pH 9.2, 10.0, 11.6, 12.2, 12.6 at 62.5, 125, 250, 500 and 1000 mg/L, respectively. Within the contact time of 3 hours, the initial pH was considerably lowered which is likely attributable to the reaction of the test item with CO2 in the medium to CaCO3. Therefore the initial pH is considered to be the main reason for the effects of the test item on the test organisms. The 3-h EC50 was 300.4 mg/L.

The toxicity to aquatic micro-organisms of calcium carbonate (nano) was assessed in a study performed according to OECD TG 209 under GLP (Youngs, 2010). Activated sewage sludge was exposed to calcium carbonate at nominal test concentrations of 0, 10, 32, 100, 320 and 1000 mg/L for 3 hours. No toxic effects were seen at any concentration of calcium carbonate (nano) tested. Hence the 3-h EC50 was >1000 mg/L and the NOEC was 1000 mg/L. Calcium carbonate (nano) is therefore not toxic to aquatic microorganisms at concentrations up to 1000 mg/L. Evidence of undissolved test material was observed in some of the test vessels suggesting that the concentrations tested exceeded the maximum solubility of calcium carbonate in water.

Based on the results of the studies performed on calcium dihydroxide and calcium carbonate, it may be concluded that the acute toxicity to aquatic microorganisms of grades of hydraulic lime (chemical) containing up to 40% calcium carbonate will be driven by the calcium dihydroxide content and that the results available for calcium dihydroxide represent the worse-case for all grades of hydraulic lime (chemical).