AAA reaction product

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-11-24 to 2004-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed in acordance to guideline with no deviations having an impact on the validity of the testing results

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): DEA/ACID anhydride reaction product

- Physical state: highly viscous amber liquid

- Composition of test material, percentage of components:
Monomers: <17-35% (9-16% diethanolamine; 2-8% tetrahydrophthalic acid; 5-11% trimellitic acid; <0.1% tetrahydrophthalic anhydride; <0.8% trimellitic anhydride)
Dimers/trimers: <24-45%
Polymers: <10-25%
Not removable water: 5-15%

- Lot/batch No.: 200501.UN2810

- Expiration date of the lot/batch: september 2003

- Stability under test conditions: stable under storage condition

- Storage condition of test material: in the refrigerator in the original container away from direct sunlight

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
NA

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

One replicate was tested for each AAA reaction product concentration.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The final nominal concentrations tested were 10, 32, 100, 320, and 1000 mg/L. Test item amounts of 4.9, 16.1, 49.6, 159.4, and 500.0 mg were weighed onto glass slides and transferred into the respective test flask. Then, 284 mL of tap water was added to each flask. The test item was mixed into the tap water using ultrasonic treatment for 5 minutes and intense stirring for one hour at room temperature. After the stirring period of one hour, the test media appeared to be clear solutions. Then, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added.

Synthetic wastewater composed on the following components in 1 L of deionized water:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 • 2H2O
0.2 g MgSO4 • 7H2O
2.8 g K2HPO4

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater per liter and was kept at room temperature under continuous aeration until use. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was 6.6.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

NA

Test conditions

Hardness:

No data

Test temperature:

20 °C

pH:

7.0 - 8.3

Dissolved oxygen:

The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before the measurements of the respiration rates, the dissolved oxygen concentrations were at least 8.0 mg/L

Salinity:

NA

Nominal and measured concentrations:

The final nominal concentrations tested were 10, 32, 100, 320, and 1000 mg/L amounting 4.9, 16.1, 49.6, 159.4, and 500.0 mg of AAA reaction product

Details on test conditions:

TEST SYSTEM
The test was performed in 1000-mL glass flasks. The test flasks were labeled with the necessary information to ensure unmistakable identification.
At the start of the test, 200 mL activated sludge inoculum with a sludge concentration of 3.0 g/L dry weight (corresponding to about 1.2 g dry material per liter test medium) was added. The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, then to the test solutions of the reference item, then to the test solutions of the test item, and finally to the second control.
During the incubation period of exactly 3 hours, all test media and the controls were continuously aerated with compressed air at a flow of approximately one liter per minute. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before the measurements of the respiration rates, the dissolved oxygen concentrations were at least 8.0 mg/L. The temperature in the test media, measured in one control, was 20 °C at the start and at the end of the incubation period.

TEST MEDIUM / WATER PARAMETERS
In accordance with the test guidelines, one replicate was tested for each test item concentration. Additionally, two controls (tap water, synthetic wastewater and activated sludge inoculum, without test item) and the reference item 3,5-dichlorophenol (positive control) at nominal concentrations of 5, 16, and 50 mg/L were tested in parallel under otherwise identical conditions of the test.

The chemical oxygen consumption of the test item was investigated in another study (28-day ready biodegradability test). Over 28 days, no oxygen consumption was determined in an abiotic control containing the test item at a nominal concentration of 100 mg/L (without activated sludge). Therefore, no abiotic control was included to measure the chemical oxygen consumption after the 3-hour incubation period.

For the dosage of the reference item, a stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5 g of 3,5-dichlorophenol was dissolved in 10 mL 1 M NaOH and diluted to about 30 mL with purified water. Excess of NaOH was neutralized with 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one liter with purified water. The pH was determined to be 7.6. The final concentration of 3,5 dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock solution were mixed with synthetic wastewater and tap water in the respective test flasks to obtain the desired concentrations.

OTHER TEST CONDITIONS
The pH-values and the dissolved oxygen concentrations were determined in all test media and the controls at the start and at the end of the 3-hour incubation period. The water temperature was measured in one control at the start and end of the incubation period, and was continuously monitored in all test media and the controls during measurement of the respiration rate. Before the addition of activated sludge, the appearance of the test media was recorded.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : For measurement of the respiration rate, a well-mixed sample of each test medium was poured into a BOD-flask after exactly three hours incubation time, and was not further aerated. Then, the dissolved oxygen concentration was measured

TEST CONCENTRATIONS
The final nominal concentrations tested were 10, 32, 100, 320, and 1000 mg/L. Test item amounts of 4.9, 16.1, 49.6, 159.4, and 500.0 mg were weighed onto glass slides and transferred into the respective test flask.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 1 000 mg/L

Effect concentrations 2

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Details on results:

Up to and including the concentration of 1000 mg/L, AAA reaction product had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. No concentration effect relationship was determined. Thus, the 3-hour EC20, EC50, and EC80 could not be calculated, but were clearly higher than 1000 mg/L. The oxygen consumption rates of the two controls (run at the start or at the end of the test) differed by 3.4% (guideline-recommended maximum variation: 15%

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 11 mg/L (the 95% confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming the suitability of the activated sludge used.

Reported statistics and error estimates:

NA

Any other information on results incl. tables

The chemical oxygen consumption of AAA reaction product was investigated in a 28-day ready biodegradability test. Over 28 days, no oxygen consumption was determined in an abiotic control containing AAA reaction product at a nominal concentration of 100 mg/L (without activated sludge). Therefore, no abiotic control was included to measure the chemical oxygen consumption after the 3-hour incubation period.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The inhibitory effect of AAA reaction product on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to OECD 209. AAA reaction product had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. The 3-hour EC20, EC50, and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

Executive summary:

The inhibitory effect of AAA reaction product on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to OECD 209. The following nominal concentrations of AAA reaction product were tested: 10, 32, 100, 320 and 1000 mg/L. Concentrations exceeding 1000 mg/L were not tested. In addition, two controls and three different concentrations (5, 16 and 50 mg/L) of 3,5‑dichlorophenol (positive control) were tested in parallel. The results of these treatments confirmed the suitability of the activated sludge and the method used.

Up to and including the concentration of 1000 mg/l, AAA reaction product had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. The 3-hour EC20, EC50, and EC80 could not be calculated, but were clearly higher than 1000 mg/L.