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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Sep - 11 Sep 2012
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
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Niedersachsen, Germany
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 weighed out directly in an Erlenmeyer flask, filled up with 200 mL demineralised water and stirred overnight.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Origin: Municipal sewage treatment plant of 31137 Hildesheim, Germany. Non adapted activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste. Date of receipt was Sept 10th, 2012
- Pretreatment: The sludge was washed twice and was used within 24 h after sampling.
- Dry sludge concentration: 3.46 g/L (corresponding to 1.73 g/L suspended solids in the test vessels)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
21 - 22 °C
pH:
pH-value of the activated sludge: 7.95
pH-value of the synthetic waste water: 7.53
Nominal and measured concentrations:
1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 500 mL Erlenmeyer flasks, DIN 12380, filled with 500 mL test medium
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70% saturation and to maintain the sludge flakes in suspension

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Composition of test medium: 250 mL inoculum, 16 mL synthetic waste water, 234 mL demineralised water


OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The oxygen uptake rates of all samples were determined following an incubation period of 3 h.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study: 4%, -5%, and -13% inhibition at 10, 100, and 1000 mg/L, respectively
Reference substance (positive control):
yes
Remarks:
copper (II) sulphate pentahydrate
Key result
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
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:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item was floating undissolved on the surface of the test medium
Results with reference substance (positive control):
EC50(3h) = 107 mg/L (95% CL = 97.7 - 118 mg/L)
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC calculations. Before running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for the Normality test was 0.05 and for the Equal Variance test <0.05. The Alpha-value (acceptable probability of incorrectly concluding that there is a difference) is Alpha=0.05. The EC-value of the reference item was calculated by linear regression using software GraphPad Prism5, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures.

Methyl laurate caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Table: Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of Control, Test and Reference Item Concentrations

Test Item and Reference Concentration [mg/L]

Repl.

Oxygen Uptake Rate R [mg O2/L x h]

Specific Respiration Rate Rs [mg O2/g x h]

Inhibition [%]

Mean Inhibition [%]

Control

1

23.2

13.4

 

 

2

(7.6)#

(4.4)#

3

23.6

13.6

4

24.0

13.9

5

22.4

12.9

6

23.6

13.6

Test item 1000

1

26.8

 

-15

-25

2

28.8

-23

3

32.0

-37

Reference item 58

1

22.8

3

11

2

21.2

9

3

18.4

21

Reference item 100

1

12.4

47

49

2

11.6

50

3

11.6

50

Reference item 180

1

3.2

86

80

2

6.8

71

3

4.0

83

# outlier, not included in calculation

Validity criteria fulfilled:
yes
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:
Category justification is attached in chapter 13.
Reason / purpose for cross-reference:
read-across source
Key result
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 on result:
other: CAS 111-82-0

Description of key information

NOEC (3 h) ≥ 1000 mg/L (nominal) for activated sludge (OECD 209, read across)

Key value for chemical safety assessment

Additional information

No data investigating the toxicity to microorganisms are available for Fatty acids, C12-14 (even numbered), methyl esters (CAS No. 308065-15-8).

 

Therefore, data from a structurally related category member, methyl laurate (CAS No. 111-82-0) are used as read-across (in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5). Both substances are fatty esters esterified with methanol, with fatty acid C-chain lengths of: C12-14 (CAS No. 308065-15-8) and C12 (CAS No. 111-82-0) respectively. The target substance has a high content of C12 fatty acids (30-100%). Furthermore, methyl laurate (C12 FA content > 80%) exhibits the highest toxicity to aquatic organisms (Daphnia and algae) throughout the whole SCAE Me category, with EC50 and NOEC values < 1 mg/L.Therefore, methyl laurate is used as worst-case read-across substance in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5.

 

The study evaluating the toxicity of methyl laurate to aquatic microorganisms (Fiebig, 2013) was conducted according to OECD Guideline 209, under GLP conditions. Activated sludge microorganisms from a municipal sewage treatment plant were exposed to the test substance for 3 hours at a concentration of 1000 mg/L (limit test). After the exposure period, no inhibiting effects of the substance on microorganisms activity were reported, leading to a NOEC (3 h) 1000 mg/L (nominal concentration).

 

In addition, Fatty acids, C12-14 (even numbered), methyl esters is poorly soluble (< 0.06 mg/L), which limits the exposure of activated sludge microorganisms in sewage treatment plants significantly. The Guidance on information requirements and chemical safety assessment, Chapter R7.b (ECHA, 2008) states that once insoluble chemicals enter a standard STP, they will be extensively removed in the primary settling tank and fat trap and thus, only limited amounts will get in contact with activated sludge organisms. Furthermore, Fatty acids, C12-14 (even numbered), methyl esters is considered to be readily biodegradable (78% and 83% in 28 days). According to Chapter R7.b of the Guidance on information requirements and chemical safety assessment (ECHA, 2008), for substances with a good biodegradation rate in a ready biodegradation test, inhibitory effects to aquatic microorganisms are not expected and the concentration tested can potentially be used as a NOEC for hazard assessment.

 

The lack of toxicity observed for methyl laurate (showing the highest aquatic toxicity within the category) confirms that the members of the SCAE Me category are not expected to show toxicity to aquatic microorganisms. This statement is supported by the readily biodegradable nature of these substances.

 

Based on the ready biodegradability of the substance and the results of the respiration inhibition test conducted with a structurally related category member (in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5), no toxicity to aquatic microorganisms is expected for Fatty acids, C12-14 (even numbered), methyl esters (CAS No. 308065-15-8).