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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-11 to 2012-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing of the stock solution in an Erlenmeyer flask, addition of 200 mL demineralised water, overnight stirring
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: Limit concentration of 1000 mg/L
- Controls: Triplicates at the beginning of the test + triplicates at the end of the test
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: Washed twice with autoclaved tap water and an adjustment to a dry weight concentration of 3.0 g/L +/- 10 % was not necessary. The sludge was used within 24 h after sampling
- Dry sludge concentration: 2.72 g/L, corresponding to 1.36 g/L in the test vessel
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
20.9 °C
pH:
pH-value of the activated sludge 7.72
pH-value of the synthetic waste water 7.44
Salinity:
Not determined
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL demineralised water


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test concentration
- Range finding study
Inhibition of Respiration (Preliminary Test)
Nominal Test Item
Concentration
[mg/L] Pretreatment pH(test item in water) pH(test item in water) Inhibition

[%]
10 Stirring overnight - - -4
100 - - -10
1000 - - -25
1000 6.82 7.24 -17






Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
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:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 99.1 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. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

From the oxygen depletion the oxygen uptake rates were calculated in mg O2/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in
mg O2/g
×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control and the Limit Test Item Concentration

Test Item Concentration

[mg/L]


Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]

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


Inhibition

[%]

Mean Inhibition

[%]

Control

1

18.4

13.5

2

18.8

13.8

3

18.8

13.8

4

20.0

14.7

5

18.8

13.8

6

17.6

12.9

1000

1

21.6

-16

-21

2

23.2

-24

3

22.8

-22

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

Reference Item Concentration

[mg/L]


Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]

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


Inhibition

[%]

Mean Inhibition

[%]

Control

1

20.0

14.7

2

18.8

13.8

3

17.6

12.9

4

18.4

13.5

5

17.2

12.6

6

16.0

11.8

58

1

12.8

29

24

2

14.0

22

3

14.0

22

100

1

9.2

49

50

2

8.4

53

3

9.2

49

180

1

3.6

80

79

2

4.0

78

3

4.0

78

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item Glyceryl Stearate Citrate (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) is 1000 mg/L.
The test item is not toxic at the limit concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Glyceryl Stearate Citrate (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) (batch number: 105618) from 2012-06-11 to 2012-06-12, with the definitive experimental phase on 2012-06-12 at the Dr. U.Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit test item concentration of 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -16 % to -22 %.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 99.1 mg/L.

Table 1: NOEC-Value of Glyceryl StearateCitrate (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts)

Test Item Concentration
[mg/L]

NOEC

1000

                                              

The NOEC of the test item Glyceryl Stearate Citrate (Glycerides, C16-18 mono-, di-, and tri-, hydrogenated, citrates, potassium salts) is 1000 mg/L.

The test item is not toxic at the limit concentration of 1000 mg/L to activated sludge
of a municipal sewage treatment plant.

Description of key information

NOEC (3 h): 1000 mg/L (OECD 209); read-across

Key value for chemical safety assessment

Additional information

Since no studies investigating the toxicity of Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates (CAS 91052-16-3) to activated sludge microorganisms are available for this endpoint, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read across to the structurally related source substance Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6) was conducted. The target substance Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates (CAS 91052-16-3) and the source substance Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6) are considered to be structurally very similar. The difference between both substances is that the source substance is the potassium salt of the target substance.

 Since both substances share common similar functional groups and neither the target nor the source substance consist of any structural alerts leading to a different toxicity profile this read-across is suitable. This read-across is further justified within the analogue justification in IUCLID Section 13. In this case of read-across, the best suited (highest degree of structural similarity, nearest physico-chemical properties) source substance was used for the assessment.

The study available for the read-across substance Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6) was performed according to OECD 209 (Fiebig, 2012). Activated sludge from a domestic sewage treatment plant was used for testing and incubated for 3 h. The respiration rate was recorded after incubation and no inhibition of respiration was recorded. Instead a stimulation of respiration was measured. Thus, a NOEC (3 h) of 1000 mg/L was derived.

Based on the available result from one structurally related source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which is characterized by a comparable structure, it can be concluded that Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates will not exhibit inhibit the degradation process in commercial sewage treatment plants.