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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May 2017 - 16 Jun 2017
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 (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the dark brown testbottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item ¿ Milli-RO water mixtures were magnetically stirred for a short period of 4 to 11 minutes. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sewage sludge was sourced from a municipal sewage treatment plant ('Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands) which predominantly receives domestic sewage.

The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.4 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
not specified
Water media type:
other: Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Total exposure duration was 3 hours, during which aeration and stirring took place.
Post exposure observation period:
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
Hardness:
Not relevant
Test temperature:
19 to 21°C
pH:
7.4 to 7.6 (before addition of sludge), 7.1 to 8.0 (after the 3 hour exposure period)
Dissolved oxygen:
60-70% saturation (60% of air saturation is >5 mg/L at 20°C)
Salinity:
Not relevant
Conductivity:
Not relevant
Nominal and measured concentrations:
Limit/range finding test loading rates: 10, 100 and 1000 mg/L (highest loading rate tested in triplicate, one replicate per lower loading rates).
Final test loading rates: 100, 180, 320, 560 and 1000 mg/L (five replicates per test group, six replicates for the control).
Details on test conditions:
Aeration of test vessels was adjusted so that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.

The pH of all test vessels was determined before addition of sludge and after the 3 hour contact time in the remaining part of the reaction mixture.

The medium temperature was recorded continuously in temperature control vessels during the test. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: ELR50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
other: NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Limit/range finding tests showed no inhibition of respiration rate at 10 and 100 mg/L loading rates and 10% inhibition of respiration rate at a loading rate of 1000 mg/L. There was no oxygen uptake from abiotic processes. All test conditions and acceptability criteria prescribed by the study plan were met.

Final test results showed that no statistically significant inhibition of respiration rate of the sludge was observed at any of the tested loading rates. Thus, the NOELR was concluded to be 1000 mg/L and the ELR50 was concluded to be >1000 mg/L.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was 2.57 mg/L and was therefore in the accepted range of 2 to 25 mg/L for total respiration.

Final Test ¿ Overview of the Results

Treatment

Loading rate (mg/L)

Mean respiration rate

% Inhibition of the respiration rate (mean value)

(mg O2/L h)

(mg O2/g h)1

Control

 

33.18

22.12

 

T1

100

32.07

21.38

3.37

T2

180

35.28

23.52

-6.31

T3

320

33.72

22.48

-1.63

T4

560

35.75

23.84

-7.75

T5

1000

33.82

22.55

-1.92

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

Validity criteria fulfilled:
yes
Conclusions:
Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol is not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR). The ELR50 was concluded to be >1000 mg/L.
Executive summary:

The influence of Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol on the respiration rate of activated sludge was investigated after a contact time of 3 hours was investigated according to OECD Guideline 209.

 

Due to insufficient solubility of the test item, test solutions were prepared by adding weighed amounts of test item into test bottles containing 200 mL Milli-RO water, and magnetically stirred for 4 to 11 minutes. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Five loading rates of the test item were tested, ranging from 100 to 1000 mg/L.

 

Results showed no statistically significant inhibition of the respiration rate of activated sewage sludge at any of the tested loading rates. It is therefore concluded that Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol was not toxic to activated sludge bacteria at or below a loading rate of 1000 mg/L. A NOELR of 1000 mg/L and a ELR50 of >1000 mg/L were derived. 

Description of key information

The influence of Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol on the respiration rate of activated sludge was investigated after a contact time of 3 hours was investigated according to OECD Guideline 209. Results showed no statistically significant inhibition of the respiration rate of activated sewage sludge at any of the tested loading rates. It is therefore concluded that Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2 -dicarboxylic anhydride and propylidenetrimethanol was not toxic to activated sludge bacteria at or below a loading rate of 1000 mg/L. a NOELR of 1000 mg/L and a ELR50 of >1000 mg/L were derived (Bachelor, 2017).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information