Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

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:

2017-06-06 to 2017-08-02

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))

Version / remarks:

adopted July 22, 2010. (Limit Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 50 mg of test item were directly weight into the test vessels and test water was added to obtain a final concentration of 100 mg/L. The composition was stirred intensively.
- Concentration: (nominal) 100 mg/L (three replicates)
- Differential loading: Limit test 100 mg/L (nominal)
- Controls: Six controls (pure water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.
PREPARATION OF REFERENCE ITEM SOLUTION: 3,5-Dichlorophenol/L
- Stock solution concentration: (according to the OECD Guideline No. 209) 0.5 g of 3,5-dichlorophenol was dissolved in 500 mL pure water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it has cooled to room temperature. The final pH was determined to be 7.0. For the adjustment of the pH 1 M NaOH was used.
- Concentrations: (nominal) 1, 4, 16 mg/L

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.
- Conditioning: The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (30 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage (see below) per litre and kept aerated at room temperature overnight.
- Initial biomass concentration: 3 g/L; This level gave a concentration of 1.5 g/L suspended solids in the test medium.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gave a concentration of 1.5 g/L suspended solids in the test medium.

- pH of the activated sludge: 6.1; no adjustment was necessary

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20°C ± 2°C

pH:

test item: (at start) 7.1, (at the end) 7.6
reference item: (at start) 7.2, (at the end) 8.1 -8.2
control: (at start) 7.1 - 7.2, at the end) 7.6 - 8.1

Dissolved oxygen:

Oxygen concentration [mg O2/L]
test item: (start) 6.8 - 8.8, (end) 7.2 - 7.6
reference item: (start) 8.3 - 8.8, (end) 7.9 - 8.2
control: (start) 7.6 - 8.8 (end) 7.4 - 8.1

Nominal and measured concentrations:

test item: 0, (nominal) 100 mg/L
reference item: (nominal) 1, 4, 16 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks, Karlsruher flasks
- Material, size, headspace, fill volume:approximately 1 L (glass flasks), 250 mL (Karlsruher flasks)
- Aeration:Compressed air (1.017 litre per minute)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per positive control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.5 g/L (suspended solids in the test medium)
- Nutrients provided for bacteria: yes, synthetic sewage feed, 16 mL added to each test vessel
(1 L synthetic sewage feed contains: 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 per litre deionised water)
- Nitrification inhibitor used (delete if not applicable): none


OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Details on termination of incubation: For the measurement of the respiration rate a well-mixed sample of test medium from each flask was poured into a Karlsruher flask after exactly 3 hours incubation time and was not further aerated during measurement. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate (oxygen consumption)
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test
- Test concentration: 100 mg/L
Preparation of test flasks: see table 1 in Any other informationon on materials and methods incl. tables.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

mean respiration rate of activated sludge treated with the test item at a concentration of 100 mg/L differed from the mean respiration rate in the control by less than 10%.

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Blank controls oxygen uptake rate: The oxygen uptake in the blank controls was 19.6 mg oxygen/g/h.
- Coefficient of variation of oxygen uptake rate in control replicates: The respiration rates of the six controls did not differ by more than 30%. The coefficient of variation was 6.4%.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 3-hour EC10: 0.5 mg/L (95%-C.I.: 0.1 - 1.9)
- 3-hour EC20: 1.0 mg/L (95%-C.I.: 0.3 - 3.4)
- 3-hour EC50: 5.0 mg/L (95%-C.I.: 1.1 - 21.7) (thus in the range of 2 to 25 mg/L for the activated sludge batch which was used)

Reported statistics and error estimates:

The software used to perform the statistical analysis was ToxRat Professional®, Version 3.2.1, ToxRat Solutions GmbH.
The EC50 including the 95 %-confidence limits of the reference item was calculated by Non-linear Regression, using the Levenberg Marquardt Optimization and the Monte-Carlo simulation for calculation of confidential limits. For the test item, the Shapiro-Wilks Test (α=0.01) on normal distribution and the Levene´s Test (α=0.01) on variance homogeneity. Difference between test item concentration and control was tested by Student t-test for homogenous variances (α=0.05).

Toxicity of Test Item

Inhibition of Respiration Rate: The mean respiration rates of the activated sludge treated with the test item at a concentration of 100 mg/L differed by less than 10% from control.

Conclusion: At a nominal test item concentration of 100 mg test item/L less than 10% inhibition was observed after three hours of incubation. The mean deviation in the respiration rate between VOELOFA Monomer (n=3) and the control (n=6) was -6.9%.

The respiration rate of the activated sludge with VOELOFA Monomer at a test item concentration of 100 mg/L was not statistically significant reduced when compared to the control (Student t-test). VOELOFA Monomer at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge. Therefore, it can be assumed that the NOEC is above 100 mg test item/L.

Validity Criteria of the Study

Inoculum Control: The respiration rates of the six controls did not differ by more than 30%. The coefficient of variation was 6.4%. The oxygen uptake in the blank controls was 19.6 mg oxygen/g/h. Reference Item: The 3-hour EC50 of the reference item 3,5-Dichlorophenol was determined to be 5.0 mg/L and was thus in the range of 2 to 25 mg/L for the activated sludge batch which was used.

Validity criteria fulfilled:

yes

Conclusions:

VOELOFA Monomer at the rate of 100 mg/L had no toxic effects on respiration activity of activated sludge. Therefore, it can be assumed that the NOEC is above 100 mg/L.

Executive summary:

The influence of VOELOFA Monomer on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study was conducted according to the OECD test guideline 209 (July 22nd, 2010).

A concentration of 100 mg/L was tested. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring.

No significant inhibition of respiration rate of the sludge was recorded at 100 mg VOELOFA Monomer per litre. No further testing was needed.

Since all criteria for acceptability of the test were met, this study was considered to be valid:

> The coefficient of variation (CV) of the blank respiration rates was less than 30 %.

> The blank controls oxygen uptake rate was 19.6 mg oxygen per one gram of activated sludge per hour.

> The EC50 of the reference item was within the acceptable range of 2 to 25 mg/L.

In conclusion, under the conditions of the present test, VOELOFA Monomerw as not toxic to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/L.

Description of key information

In an Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)according to the OECD guideline 209 the toxicity of the test item towards microorganisms was tested. No toxic effect was observed up to 100 mg/L.

NOEC > 100 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L