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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March 2017 - 28 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECDTG301B
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
TEST ITEM INFORMATION
Test item: 207811/A
Identification: Soybean oil, epoxidized, Me ester, reaction products with propylene glycol
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Batch: EM97160711
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions: until 20 July 2017 (expiry date)
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Analytical monitoring:
yes
Remarks:
Analysis of CO2 produced
Details on sampling:
Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
Vehicle:
no
Details on test solutions:
TEST CONCENTRATION AND PREPARATION OF TEST SOLUTIONS
On the day of testing, weighed amounts of the test item were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test item bottle A: 33.93 mg; test item bottle B: 33.94 mg and toxicity control bottle: 34.17 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Treatment:
The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (31 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
Test type:
static
Water media type:
freshwater
Total exposure duration:
28 d
Test temperature:
22.2 -23.7°C
pH:
7.4 - 7.8
Nominal and measured concentrations:
17 mg/L test substance, coorespnding to 12 mg/L TOC
Details on test conditions:
Test duration
28 days for the inoculum blank and test item (last CO2 measurement on day 29).
14 days for the positive and toxicity control (last CO2 measurement on day 15).
During the test period, the test media were aerated and stirred continuously.
Test vessels: 2 litre brown coloured glass bottles.
Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
Stock solutions of mineral components
A)
8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
Mineral medium: 1 litre mineral medium contains:
10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm):
A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination: The test media were excluded from light
Reference substance (positive control):
yes
Remarks:
sodium acetate
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: Based on significant biodegradation of the test substance at 28d and > 25% biodegradation in the toxicity control at day 14
Details on results:
No evidence of toxicity of the test substance to the inoculum was noted at any point in time.
In the toxicity control, more than 25% biodegradation occurred within 14 days (51%, based on ThCO2).
Therefore, the test item was assumed not to inhibit microbial activity.
Results with reference substance (positive control):
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve

CO2 production and percentage biodegradation of the control and test item (bottle A & B).

Day  

Blank

Bottle A

Bottle B

 

CO2cumulative (mg)

CO2cumulative (mg)

Degradation (%)1

CO2cumulative (mg)

Degradation (%)1

1

4

6

8

11

15

19

22

25

292

292

292

2.2

7.5

11.2

14.6

19.3

23.6

29.1

33.7

37.9

43.8

46.6

47.8

0.0

2.4

8.5

15.8

21.8

27.1

30.6

33.3

36.3

38.9

40.5

40.5

0

3

10

18

25

31

35

38

42

44

46

46

0

1.9

8.1

15.6

22.2

28.0

32.2

34.9

38.1

39.7

40.7

42.5

0

2

9

18

25

32

37

40

43

45

47

48

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 87.6 mg CO2/2L.

2): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by

addition of HCl.

CO2production and percentage biodegradation of the toxicity control.

Day

HCl (0.05 N) titrated (mL)

Produced CO2

(mL HCl)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)

(%)

Blank

(mean)

Toxicity control

1

49.14

50.00

0.00

0.0

0.0

0

4

46.08

29.84

16.24

17.9

17.9

10

6

47.20

33.11

14.09

15.5

33.4

19

8

48.12

35.35

12.77

14.0

47.4

27

11

47.06

30.64

16.42

18.1

65.5

38

152)

47.06

26.20

20.86

22.9

88.4

51

1): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and positive control: 173.7 mg CO2/2L (ThCO2test item: 88.2 mg CO2/2L + ThCO2sodium acetate: 85.5 mg CO2/2L)

2): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of a ready biodegradability screening test (OECD TG301B), the substance is considered not to inhibit micro-organisms, when dosed at 17 mg/L test substance.
Executive summary:

Toxicity to microorganisms of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was investigated in a study conducted in accordance with OECDTG 301B (carbon dioxide evolution test (modified Sturm test)) and GLP. In this study activated sludge was exposed to 17 mg/L, corresponding to 12 mg TOC/L, of the test substance for 28 days. The validity criteria of the test were met. The relative biodegradation values calculated from the measurements performed during the test period revealed significant (46% and 48%) biodegradation of the test substance (based on ThCO2), for the duplicate bottles tested. In the toxicity control, the test substance was found not to inhibit microbial activity (51% biodegradation in 14d). Therefore it is concluded that Soybean oil, epoxidized, Me ester, reaction products with propylene glycol has no toxicity to micro-organisms at the tested conc of 17 mg/L and this is considered the NOEC for micro-organisms.

Description of key information

Toxicity to microorganisms of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was investigated in a study conducted in accordance with OECDTG 301B (carbon dioxide evolution test (modified Sturm test)) and GLP. In this study activated sludge was exposed to 17 mg/L, corresponding to 12 mg TOC/L, of the test substance for 28 days. The validity criteria of the test were met. The relative biodegradation values calculated from the measurements performed during the test period revealed significant (46% and 48%) biodegradation of the test substance (based on ThCO2), for the duplicate bottles tested. In the toxicity control, the test substance was found not to inhibit microbial activity (51% biodegradation in 14d). Therefore it is concluded that Soybean oil, epoxidized, Me ester, reaction products with propylene glycol has no toxicity to micro-organisms at the tested conc of 17 mg/L and this is considered the NOEC for micro-organisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
17 mg/L

Additional information