2-Hydroxyethylmethacrylate, reaction products with proyleneoxide and Phthalic anhydride

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "The Guidelines for the Testing of Chemicals, Effects on Biotic Systems" (the 2nd edition), Beijing: China Environment Press, 2013

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Chemicals-Activated Sludge, Respiration Inhibition Test (GB/T 21796-2008)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

Test guideline 6

Qualifier:

according to guideline

Guideline:

other: The Guidelines for the Testing of Chemicals (HJ/T 153-2004)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Purity: >99%

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Remarks:

Deionized water

Details on test solutions:

Preparation of test solutions: A reference control stock solution of 3,5-dichlorophenol (DCP), a known inhibitor of microbial respiration was prepared as follows: 1.0009 g of 3,5-dichlorophenol was dissolved in 20 mL of 1 mol/L NaOH, diluted to approximately 60 mL with deionized water. While stirring, 0.5 mol/L H2SO4 was added to the point of incipient precipitation, approximately 16 mL of 0.5 mol/L H2SO4 was required. Finally, the mixture was diluted to 1 L with deionized water. The pH was 7.78 and adjusted with 0.5 mol/L H2SO4.
A stock solution of test substance at 1130 mg/L: 0.1130 g test substance was solute and made up to 100 mL with deionized water.

Range-finding test: 11 beakers of 1000 mL were prepared. At time 0 min, 0.5429 g test substance was added to the first vessel with 484 mL deionized water (Abiotic control RA), then 16 mL of the synthetic sewage feed was added. Final volume of the test solution was 500 mL. The mixture was aerated at 0.8 L/minute using a Pasteur-pipette as an aeration device. At time 15 min, 16 mL of the synthetic sewage feed and 250 mL of microbial inoculum were added to the second vessel (first blank control RB1), then made to final volume of 500 mL with deionized water. The mixture was aerated at 0.8 L/minute using a Pasteur-pipette as an aeration device. At time 30 min, the above procedure was repeated, except that 0.0054 mg test substance was added, then 16 mL of synthetic sewage and 250 mL microbial inoculum and appropriate volume of deionized water was added successively to make a total volume of 500 mL (Test RT1-5). This process was repeated at 15-minute intervals with different amounts of the test substance to give a series of vessels containing widely-spaced concentrations of the test substance.

In the range-finding test, the test solutions of the test substance with nominal concentration 10, 100, 1000 mg/L were established, with no replicates for tested concentrations 10 and 100 mg/L, but triplicates at the highest tested concentration 1000 mg/L.

The reference control (RR1-3) was tested on the microbial inoculums in the same way. 1.50, 5.0, 15.0 mL stock solution of reference substance were added to test bottles respectively with exposure solutions at 3, 10, and 30 mg/L. Finally, a second negative control (RB2) was prepared. Air temperature and pH of all test solutions were measured at test start and test end.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Activated sludge microorganisms were obtained from a Chengdong waste water treatment plant receiving predominantly domestic sewage. On arrival at the laboratory, the sludge was washed three times with deionized water using the following method: After centrifuging at approximately 2500 rpm for 10 minutes, the supernatant was decanted and the remaining sludge solids re-suspended in deionized water and mixed well with mechanical stirrer. After the third spin, the wet solids were homogenized for 2 minutes in a blender. A small amount of the fresh sludge was weighed, oven-dried, and reweighed. A calculation was made from these results to determine the amount of wet sludge that must be suspended in deionized water in order to obtain an activated sludge with a mixed-liquor suspended solids (MLAA) level of approximately 3 g/L. Then appropriate amount of solids equal to 150 g wet solids (containing 90% water) were suspended in 5 L of deionized water and 250 mL of the synthetic feed stock solutions. The sludge suspension was aerated overnight at 20°C ± 2°C. The pH of the overnight (16 hours) suspension was 7.35. Viability of the microorganisms was checked by means of a reference control.

Study design

Water media type:

other: Deionized water

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

19.6-21.4°C

pH:

7.35-7.84

Dissolved oxygen:

2 mg/L

Nominal and measured concentrations:

10, 100, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L beaker
- Type: Open
- Aeration: Clean, oil-free air. 0.8 L/min
- No. of vessels per concentration: Triplicates only at high concentration
- Synthetic sewage food: Made by dissolving the following amounts of substances in 1 L of deionized water: 16 g peptone, 11 g meat extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2.H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4.

- Range finding study: The result of range-finding test showed that the 3 h EC50 of the test substance was greater than 1000 mg/L, so there was no need to conduct the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Key result

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

Details on results:

The result indicated that no obvious oxygen uptake had taken place in the abiotic control since the oxygen concentration was 7.60 and 7.58 mg O2/L at the beginning and at the end of the test. EC50 values were calculated by regression analysis and the result show that 3 h EC50 value of the reference control, 3,5-DCP was 5.87 mg/L, and the 95% confidence limit ranged from 4.90 to 7.00 mg/L, which was in the range of 2 to 25 mg/L. The oxygen uptake rates of negative controls were 33.1 and 37.4 mg O2/g.h, which was not less than 20 mg O2/g.h. The coefficient of variation of oxygen uptake rate in control replicates was 12.1%, which was not more than 30%. The validity criteria were fulfilled.

Results with reference substance (positive control):

3 h EC50: 5.87 mg/L (95% confidence limit: 4.90-7.00 mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

3 h EC50: > 1000 mg/L

Executive summary:

A 3-hour activated sludge respiration inhibition test was performed using activated sludge from a domestic waste treatement plant according to "The guidelines for the testing of chemical (HJ/T 153-2004)" and with reference to OECD TG 209.

In the range-finding test, the sludge microorganisms were exposed to three concentrations of the test substance at nominal concentrations of 10, 100, and 1000 mg/L. The respiration rate, expressed as oxygen consumption by the microbes in mg O2 per liter per hour, was measured under defined conditions following the 3-hour exposure period. Inhibition values were calculated by comparing test respiration rates to negative control rates.

EC50 values were calculated by regression analysis. The result indicated that no obvious oxygen uptake had taken place in the abiotic control since the oxygen concentration was 7.60 and 7.58 mg O2/L at the beginning and at the end of the test. The 3 h EC50 value of the reference control, 3,5-DCP was 5.87 mg/L, and the 95% confidence limit ranged from 4.90 to 7.00 mg/L, which was in the range of 2 to 25 mg/L. The oxygen uptake rates of negative controls were 33.1 and 37.4 mg O2/g.h, which was not less than 20 mg O2/g.h. The coefficient of variation of oxygen uptake rate in control replicates was 12.1%, which was not more than 30%. The validity criteria were fulfilled.

Under the above valid test conditions, the 3-hour EC50 value of the test substance was greater than 1000 mg/L.