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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Mar 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted in 1984
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was directly added to the test medium.
Test organisms (species):
activated sludge
Details on inoculum:
- Source of inoculum: Activated sludge was collected from the sewage treatment plant of Therwil, Switzerland on 06 Mar 1989.
- Preparation of inoculum for exposure: The sludge was separated from the aqueous layer by settling.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
18 - 20 °C
Nominal and measured concentrations:
Nominal: 0, 1.1, 3.3, 10.7, 32.0, 100.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: BOD flasks with gas inlet
- Size, fill volume: 250 mL, 200 mL
- Aeration: Yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Concentration of sludge: 1.6 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated drinking water

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Microbial respiration was measured after 3 h of incubation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 3.2
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
other: IC20
Effect conc.:
75 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:
IC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Validity criteria are fulfilled.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: IC50 = 13 mg/L

Table 1: Inhibition of microbial respiration

Concentration (mg/L)

Consumption Rate (mg/L*h)

Inhibition (%)

Blank 1

40.62

2

Blank 2

42.21

-2

1.1

41.62

0

3.3

43.97

-6

10.7

41.79

-1

32.0

35.70

14

100.2

30.00

28

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Sep 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
Version / remarks:
adopted in 1984
Deviations:
yes
Remarks:
Higher temperature during the test (Guideline: 18 - 20 °C, Test: 23.3 - 24.4 °C)
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
adopted in 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 10 g/L was prepared using the following procedure: 10 g test item was mixed with 1.1 g NaOH (45%). 40 mL deionised water (80 – 100 °C) was added. After homogenisation with a glass stirrer, 85 mL deionised water (room temperature) was added. Then, the vessel was stirred on a magnetic stirrer. 0.5 M NaOH was added until the resulting solution was clear. The pH of the solution was adjusted to 7.8 ± 0.2 with 1 M HCl. the solution was transferred into a volumetric flask and filled up to 1000 mL with deionised water.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of the inoculum: The sludge was taken from the activation basin of the sewage plant in Edenkoben, Germany
- Laboratory culture: Upon arrival, the sludge was washed with tap water three times, centrifuged and resuspended in tap water.
- Suspended solids: 4.2 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
pH:
7.7 - 8.1
Nominal and measured concentrations:
Nominal: 0, 1.0, 3.2, 10, 32, 100, 320, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Beakers
- Material, fill volume: Glass, 500 mL
- Aeration: Purified air at 0.75 L/min
- No. of vessels per concentration (replicates): 2 for treatment 1000 mg/L, 1 for all other treatments and positive control
- Concentration of suspended solids: 1.7 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: < 1.0 mg/L
- Metals (mg/L): Na 4.7, K 2.6, Ca 33.1, Mg 2.1, other metals < 0.0005 to < 0.01
- Pesticides: < LOD
- Chloride: 9.0 mg/L
- Conductivity: 189 µS/cm
- Intervals of water quality measurement: pH was measured at test end.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Respiration rate was measured after 3 h of incubation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
130 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 62 - 310 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 480 - 7200 mg/L; extrapolated
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- The inhibition caused by the test item did not rise above 45%. Therefore the EC50(3h) was determined by extrapolation.
- All validity criteria are fulfilled.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 9.1 mg/L, 95% CL = 7.2 - 11.0 mg/L

Table 1: Inhibition (%) of microbial respiration after 3 h of incubation

Concentration

% Inhibition

Control

-

1.0

-3.2

3.2

1.2

10

1.7

32

16.9

100

25.5

320

24.0

1000 (1)

43.8

1000 (2)

45.3

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Sep - 05 Okt 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 1984
Deviations:
yes
Remarks:
Test temperature exceeded maximum guideline value (guideline: 20 ± 2 °C; pre-test: 21.5 - 26.1 °C; main test: 22.5 - 24.5 °C)
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
adopted 1988
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Before each experiment a solution at a nominal concentration of 10,000 mg/L in drinking water was prepared.




Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum: Activated sludge was taken from the waste water treatment plant of Neustadt/Weinstraße, Germany. The treatment plant primarily processed domestic sewage and was in proper operational order.
- Preparation of inoculum for exposure: The sludge was filtered and resuspended in drinking water. The sludge was mixed with water to give a dry weight of 4.0 ± 0.4 g/L and ventilated until ready for use in the test. The pH of the solution was measured before the test started and, where necessary, adjusted to 6.0-8.0.




Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
0.943 mmol/l
Test temperature:
20 ± 2°C
pH:
pH of sludge in main test: 7.4
Dissolved oxygen:
After 3 hours the content of the first vessel was poured into a 250 ml narrow-neck flask and the respiratory activity determined over a period of 5 minutes through the continual monitoring of oxygen concentrations.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal: 0, 10, 32, 100, 320 and 1001 mg/L
Details on test conditions:
TEST SYSTEM
TEST VESSELS:
Glass beakers with a nominal volume of 800-1000 ml were used. Flat-bottomed narrow-neck flasks with a nominal volume of 250 ml were used for the purposes of measuring oxygen levels.

IMPLEMENTATION OF TEST
Preparations:
The culture medium was freshly prepared. The inoculum was collected from the water treatment plant and washed before the dry residue was calculated.
The master solutions of the test and reference materials were produced.

Experimental Parameters:
Incubation Period: 3 hours
Replicate Samples: 2 replicates/test, 2 controls (control experiment)
Dilution Water: Drinking Water
Aeration: Continuous with purified air through Pasteur pipettes.
Flow rate of approx. 0.75 l/min.
Nutrients: Culture Medium, 32 ml/l
Temperature 20 ± 2°C

Procedure:
In the first control experiment, 16 ml of culture solution was mixed with 284 ml drinking water to which 200 ml of inoculum was added. The mixture was then aerated. Five minutes later the second control experiment was carried out in the same manner. The first reference experiment was carried out five minutes later. This involved mixing 16 ml culture solution with 1.25 ml of master solution to which drinking water was added to make up a volume of 300 ml. Next, 200 ml of inoculum was added and the mixture was aerated. The other reference, test and control experiments were carried out in an identical manner at 5 minute intervals.
After 3 hours the content of the first vessel was poured into a 250 ml narrow-neck flask and the respiratory activity determined over a period of 5 minutes through the continual monitoring of oxygen concentrations. The other vessels were tested in an identical manner at intervals of 5 minutes.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Drinking water

Synthetic Culture Medium:
Peptone 16.0 g
Meat Extract 11.0 g
Urea 3.0 g
NaCl 0.7 g
CaCl2*2H2O 0.4 g
MgSO4*7H2O 0.2 g
K2HPO4 2.8 g
H2O demineralised ad 1000 ml

The pH value of the solution was adjusted to 7.5 ± 0.5.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
After 3 hours the content of the first vessel was poured into a 250 ml narrow-neck flask and the respiratory activity determined over a period of 5 minutes through the continual monitoring of oxygen concentrations. The other vessels were tested in an identical manner at intervals of 5 minutes.

TEST CONCENTRATIONS
- Range finding study: A preliminary test was conducted.
- Test concentrations: In the preliminary test the following concentrations were used: 1000 mg/L, 100 mg/L, 10 mg/L and 1 mg/L
- Results used to determine the conditions for the definitive study: Respiratory inhibition of -1.5, 1.4 and 34% at 1, 10 and 1011 mg/L, respectively.
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
Details on results:
- See attached background material for results tables for preliminary and main study.
- Respiratory inhibition of -4, -6, 0, 3 and 18% at 10, 32, 100, 320 and 1001 mg/L, respectively.

Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (3 h): 7.0 mg/L (95% CL = 4.7 - 9.6 mg/L)

Results

The EC50 values for the reference material were determined graphically. When applying the values in a Gaussian logarithmic diagram the average square lines with the X axis parallel at 50% inhibition was used.

 

EC50 Values

Parameter

Value

95%-Confidence Range

EC50 Test material - Perlastan L-30

> 1000 mg/l

not known

EC50 Reference Material (Preliminary Test)

6.2 mg/l

4.5-7.9 mg/l

EC50 Reference Material (Main Test)

7.0 mg/l

4.7 – 9.6 mg/l

 

 

Validity

The following table presents the validity criteria used.

 

Validity Criteria for Test

Condition

Result

5 mg/l < EC50 Reference < 30 mg/l

Preliminary Test: 6.2 mg/l MainTest: 7.0 mg/l

Variation of Control Samples < 15%

Preliminary Test: max. 10 % Main test: max. 12 %

 

 

Discussion

All validity criteria were met.

The inhibition values calculated for the reference sample display good levels of agreement in both the preliminary and main tests.

The inhibition values for the test material deviated between the preliminary and main tests. As the tests involved a biological system, deviations of this nature are not unusual and all values are within the same range.

Validity criteria fulfilled:
yes
Conclusions:
The EC50 of the test material was determined to be >1000 mg/l.
Executive summary:

Two valid tests were carried out.

In the preliminary test, the following concentrations were used: 1000  mg/l, 100  mg/l, 10  mg/l and 1 mg/l. The test was carried out using activated sludge from a municipal water treatment facility. The sludge was washed before use and the dry residue was determined to be 4.23 g/l. The concentration of suspended solids used in the experiment was 1692 mg/l.

The effect on the respiratory activity of the sludge was measured after an incubation period of 3 hours by measuring the drop in oxygen levels in the deposits over a period of 5 minutes.

The reference material was 3.5‑Dichlorophenol for which an EC50 of 6.2 mg/l (95% confidence range: 4.5-7.9 mg/l) was calculated.

At a concentration of 1000 mg/l the test material produced a 34% level of inhibition. A concentration of 100 mg/l could not be evaluated. At concentrations of 10 mg/l and 1 mg/l the test material did not exhibit any inhibitory effect.

The main test was conducted with five concentrations between 10-1000 mg/l. The experiment involved the use of activated sludge from a municipal water treatment facility. The sludge was washed before use and the dry residue was determined to be 3.98 g/l. The concentration of suspended solids used in the experiment was 1592 mg/l.

The effect on the respiratory activity of the sludge was measured after an incubation period of 3 hours by measuring the drop in oxygen levels in the deposits over a period of 5 minutes.

The test material was 3.5‑Dichlorophenol for which an EC50 of 7.0 mg/l (95% confidence range: 4.9-9.6 mg/l) was calculated.

At a concentration of 1000 mg/l the test material produced an 18% level of inhibition. At all lower concentrations the test material did not exhibit any inhibitory effect.

The following results were obtained for the Perlastan L-30 test material:

3h EC20 ~ 100 mg/l

3 h EC50 > 1000 mg/l

NOEC = 100 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:
Please refer to the analogue approach justification provided in IUCLID 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
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
130 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 62 - 310 mg/L
Remarks:
Source: RA-A, EC 701-177-3, LAUS, 2009, activated sludge, 3 h, RL1
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 480 - 7200 mg/L; extrapolated
Remarks:
Source: RA-A, EC 701-177-3, LAUS, 2009, activated sludge, 3 h, RL1
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other:
Remarks:
Source: RA-A, EC 701-177-3, LAUS, 2009, activated sludge, 3 h, RL1
Duration:
3 h
Dose descriptor:
other: IC20
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other:
Remarks:
Source: RA-A, EC 701-177-3, Ciba Geigy, 1989, activated sludge, 3 h, RL2
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other:
Remarks:
Source: RA-A, EC 701-177-3, Ciba Geigy, 1989, activated sludge, 3 h, RL2
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
Remarks on result:
other:
Remarks:
Source: RA-A, CAS 137-16-6, LAUS, 2005, activated sludge, 3h, RL2
Conclusions:
Effect concentrations, EC50 (3h) of > 100 mg/L and a NOEC (3 h) of 10 mg/L (nominal) were determined.

Description of key information

NOEC (3 h) = 10 mg/L (nominal, OECD 209), read-across

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
10 mg/L

Additional information

No studies investigating the toxicity to microorganisms are available for Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3). Therefore, in accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across to the structurally related source substances Sodium N-lauroylsarcosinate (CAS 137-16-6), a sodium salt with a shorter carbon chain length and N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC 701-177-3), an acid with a longer carbon chain length was conducted. Thus, the toxicity to aquatic microorganisms can be filled by interpolation from the available source substances. Since dissociation of the sodium salt under environmental conditions yields the same ionic species as dissociation of the acidic form of the substance, it is not relevant which form of sarcosinate molecule (salt or acid) is used as source substance for the aquatic toxicity assessment. Based on the high degree of similarity between the structural and physico-chemical properties of the target and source substances, the source substances are considered suitable representatives for the evaluation of the toxicity to microorganisms of the target substance. The read-across approach is justified in detail within the analogue justification in IUCLID section 13.

All three studies followed OECD guideline 209 and included a valid reference control.

The key study conducted with the source substance N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC 701-177-3) was carried out under GLP conditions. Effects on the respiration rate of microorganisms were observed at concentrations of 32 mg/L and above (nominal), leading to an estimated EC50 (3 h) of 1300 mg/L (extrapolated) and a NOEC of 10 mg/L. The supporting study conducted with the source substance N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC 701-177-3) did not follow GLP standards. In this study, the IC50 (3 h) for respiration inhibition was above the highest tested concentration of 100 mg/L.

The supporting study with the source substance Sodium N-lauroylsarcosinate (CAS 137-16-6) followed GLP standards.The EC50 (3 h) was found to be > 1000 mg/L which was the highest tested concentration.

Based on the available results from two structurally related read-across substances (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which are characterized by a comparable structure, it can be concluded that Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) will exhibit comparable effects on activated sludge microorganisms.