Diallyl hexahydrophthalate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January 2018 - 13 January 2018 (Experimental Start to experimental completion)

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))

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

One study plan deviation: One bound copy of the final report will be sent to the Sponsor as per the contract, instead of a PDF as per the study plan. This deviation is considered not to adversely affect the results or study integrity.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Osaka Soda Co., Ltd. Japan
- Lot/batch No.of test material: 40201
- Expiration date of the lot/batch: 26 January 2019
- Purity test date: 26 September 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: No vehicle used, test item directly weighed intp flasks before experimental start
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, based on poor solubility of the test item in water, the required quantity of test Item was weighed directly into each individual flask just before the start of the experiment.

Analytical monitoring:

not specified

Details on sampling:

- Concentrations: Teh test was performed at 3 concentrations of the Test Item with 5 replicates per concentration: 10 mg/L, 100 mg/L and 1000 mg/L
- Sampling method: A well mixed sample of each treatment was poured into a BOD flask after approximately 3 hours incubation.
- Sample storage conditions before analysis: No sample storage specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on poor solubility of the Test Item in water, a given amount Test Item was weighed directly into each individual flask just before the start of the experiment.
The test was performed at 3 concentrations of the Test Item with 5 replicates per concentration: 10 mg/L, 100 mg/L and 1000 mg/L (TI31, TI32, TI33, TI34, TI35) were used.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Activated sludge, microorganisms from a domestic waste water treatment plant.
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
- Method of cultivation: Not specified
- Preparation of inoculum for exposure: The activated sludge was supplied by the sewage plant for domestic sewage 2 days before the start of teh experiment. Prior to use, the sludge was fed daily wtih 50 mL synthetic sewage per litre and let aerated at 20 +/-2°C until use.
- Pretreatment: The activated sludge was washed adn centrifuged and teh supernatant liquid phase was decanted. the solid material was re-suspended in chlorine-free tap water and again centrifuged. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight was determined.
Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water. The activated sludge was used directly after conditioning.
- Initial biomass concentration:3 g/L (dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.22. The activated sludge was used directly
after conditioning.

Test type:

static

Water media type:

other: Chlorine-free tap water

Remarks:

Untreated control used deionised water

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

Contact time of 3 hours as per OECD 209

Post exposure observation period:

Measurement of Respiration Rate (Dissolved Oxygen); 15 minutes post incubation. For the measurement of respiration rate a well-mixed sample of each treatment was poured into a BOD flask after approximately 3-hours incubation, and was not further aerated. The oxygen concentration was then measured for approximately 15 minutes with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.

Measurement of pH and temperature: The pH of the test mixtures was determined at the start and at the end of the incubation period in all test vessels. The temperature was measured in the laboratory with a min/max thermometer during the experiment.

Hardness:

Not specified

Test temperature:

Temperature between 20.2 - 21.4°C

pH:

pH of the activated sludge inoculum was determined to be pH 7.22. The activated sludge was used directly after conditioning.

pH range (study start) pH range (study end)
Abiotic control (1000 mg/L) 7.05 - 7.34 7.48 - 7.55
Blank control (0 mg/L) 7.0 - 7.38 7.28 – 7.87
Reference item 7.06 - 7.08 7.71 – 7.77
Test item 7.16 – 7.37 7.81 – 7.86

Dissolved oxygen:

Measurement of Respiration Rate (Dissolved Oxygen): For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after approximately 3-hours incubation, and was not further aerated. The oxygen concentration was measured for approximately 15 minutes with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.

Salinity:

Not specified

Conductivity:

For the measurement of the respiration rate a well-mixed sample of each treatment was aerated. The oxygen concentration was measured for approximately 15 minutes with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.

Nominal and measured concentrations:

Nonimal test concentrations: 10, 100 and 1000 mg/Lwere chosen to permit the determination of the approximate EC50 and to identify if further, more detailed analysis was required.

Details on test conditions:

TEST SYSTEM
- Test vessel: Appropraite glass beakers for 500 mL volume and BOD bottles wtih 300 mL volume
- Type: Open
- Material, size, headspace, fill volume: Glass beakers for 500 mL volume and BOD bottles with 300 mL volume.
- Aeration: with compressed at ca. 1 L/min
- No. of vessels per concentration (replicates): Test was performed at 3 test item concentrations with 5 replicates per concentration

- No. of vessels per control (replicates): 51-1 member of five replicates (B1, B2, B3, B4 and B5) of the untreated control group
(deionised water, synthetic sewage and inoculum, without addition of the Test Item) was tested in parallel with all of the test series.
- No. of vessels per vehicle control (replicates): 1-1 member of five replicates of the untreated control group (deionised water, synthetic sewage and inoculum, without addition of the Test Item)
- No. of vessels per abiotic control (replicates): In parallel to the study, abiotic control flasks were tested without inoculum, but with the highest concentration of the Test Item (1000 mg/L nominal concentration), under identical test conditions.

In summary, 3 Test Item concentration series and 1 abiotic control series were prepared in 5 parallels with one blank control series by series. In parallel with test mixtures 4 Reference Item concentrations without replicates were also prepared with 1 blank control.

- Sludge concentration (weight of dry solids per volume): 3 g/L, an aliquot of final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g/L (on dry weight basis).
- Weight of dry solids per volume of reaction mixture per unit of time: Not specified
- Nutrients provided for bacteria: Not specified
- Biomass loading rate: Not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
- Particulate matter: Not specified

OTHER TEST CONDITIONS
- Adjustment of pH: Not adjusted, activated sludge inoculum pH 7.22. In parallel with the Test Item, the Reference Item stock solution of 3,5-Dichlorophenol was checked and adjusted with NaOH solution (2 mol/L) to pH 7.42.
- Photoperiod: Not specified
- Light intensity: Not specified
- Details on termination of incubation: After 3 hours incubation, a well mixed sample of each treatment was poured into a BOD flask and no further aeration took place.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The oxygen concentration was measured for approximately 15 minutes with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.
The pH of the test mixtures was determined at the start and at the end of the incubation period in all test vessels. The pH data are presented in Table 2 (Section 4.). The
temperature was measured in the laboratory with a min/max thermometer during the experiment.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10-fold (10 mg/L, 100 mg/L and 1000 mg/L). OECD guideline states the test should be performed using at least three concentrations of the test substance, i.e. 10 mg/L, 100 mg/L and 1000 mg/L.
- Range finding study Not performed
- Test concentrations: 10 mg/L, 100 mg/L and 1000 mg/L
- Results used to determine the conditions for the definitive study: Respiration rate, ie the oxygen consumption of waste-water microorganisms in aerobic activated sludge, expressed as mg O2 per litre per minute or mg O2 per litre per hour .EC50 (if applicable) i.e, the calculated concentration of Test Item which results in a 50%
inhibition of the respiration rate.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (at nominal concentrations of 1, 3.2; 10 and 32 mg/L)

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:

other: No inhibition effect

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: Not specified
- Adsorption (e.g. of test material to the walls of the test container): Not specified
- Blank controls oxygen uptake rate: The average specific respiration rate (RS) of the untreated (Blank) controls (B1-B5) was 22.32 mg O2/L/h/g solid sludge.
- Coefficient of variation of oxygen uptake rate in control replicates: The variation coefficient of oxygen uptake rate in above Control replicates was 3.09%.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 of the positive control substance 3,5-dichlorophenol (3,5-DCP) was in teh required rang of 2 mg/L to 25 mg/L for total respiration.

Reported statistics and error estimates:

The 3-hour EC50 value of the Reference Item and its 95%-confidence limits were calculated by Probit analysis using TOXSTAT software. In case of the Test Item the EC50 was determined directly from the values of percentage of inhibition.

In comparison to inoculum controls the inhibition of the respiration rate in the case of activated sludge was between -3.96% and 29.65% in the examined range of 10 -1000 mg/L (nominal concentration).

The average specific respiration rate (RS) of the untreated (Blank) controls (B1-B5) was 22.32 mg O2/L/h/g solid sludge.

The variation coefficient of oxygen uptake rate in above Control replicates was 3.09%.

Validity criteria fulfilled:

yes

Remarks:

Blank controls oxygen uptake was >20 mg oxygen per/g of activated sludge in an hour. The coefficient of variation of oxygen uptake in control replicates was not >30%. The EC50 of the positive control was in the required range for total respiration.

Conclusions:

Under the conditions of this study the observed endpoint for the effect of the Test Item was the 3-hour EC50 value which was considered to be >1000 mg/L (nominal). Based on the results of this study, as the Test Item had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L (nominal), no further testing was necessary.

Description of key information

Under the conditions of this study the observed endpoint for the effect of the Test Item was the 3-hour EC50 value which was considered to be >1000 mg/L (nominal). Based on the results of this study, as the Test Item had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L (nominal), no further testing was necessary.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L