Bicyclo[2.2.1]heptanebis(methylamine)

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:

23 February 2016 to 21 April 2016

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 (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test material was sufficiently soluble in RO water to prepare an aqueous stock solution.
Therefore at test initiation 5 g (nominal) of the test material was weighed into a 2 L volumetric flask containing RO water. The pH of this solution was measured to be 11.87 and adjusted to 7.52 with concentrated HCL and 5N NaOH.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure:
A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works (Suffolk, UK), which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.
On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105 °C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L in test vessels.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

Measurements of the pH of the aqueous stock solutions of the synthetic sewage, the test and reference substances and of the sample of activated sludge (4 g/L) before the start of the test are:

Synthetic sewage: 7.48
Test material stock solution: 7.52
Reference substance stock solution: 7.32
Activated sludge: 7.55

Nominal and measured concentrations:

Nominal concentrations: 10, 30, 100, 300 and 1 000 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL bottles fitted with an oxygen probe, a sinter to deliver air and a nitrogen gas nozzle.
- Material, size, headspace, fill volume: Mixtures were prepared to give a final volume of 500 mL.
- Aeration: The prepared mixtures were aerated and stirred for three hours in a thermostatically-controlled water bath, using an aerator connected to a laboratory supply of oil-free compressed air (one litre/minute).
- No. of vessels per concentration: Four replicates per concentration
- No. of vessels per control: Four replicates
- No. of vessels per reference control: One replicate per concentration
- Sludge concentration: 200 mL sludge inoculum was used per vessel.
- Nutrients provided for bacteria: Synthetic sewage feed for activated sludge was prepared by dissolving the following in 1 L of reverse-osmosis water:
16 g/L peptone
11 g/L meat extract
3 g/L urea
0.7 g/L sodium chloride
0.4 g/L calcium chloride dihydrate
0.2 g/L magnesium sulphate heptahydrate
2.8 g/L di-potassium hydrogen phosphate
The solution was stored in the fridge for up to 5 days before use. The pH of the solution was 7.5 ± 0.5.
16 mL of synthetic sewage was added to each vessel.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used to prepare solutions of test mixtures, synthetic sewage and the reference substance was reverse osmosis (RO) water.

OTHER TEST CONDITIONS
- Details on termination of incubation: Following the exposure period, the aeration was stopped and the headspace of each bottle was flushed with nitrogen during the oxygen measurement phase. The instrument measured the amount of oxygen in the mixtures at one minute intervals for at least 15 minutes.

EFFECT PARAMETERS MEASURED: Oxygen consumption

TEST CONCENTRATIONS
- Test concentrations: 10, 30, 100, 300 and 1 000 mg/L

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

Details on results:

The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 22.9 mgO2/gh with a coefficient of variation (CV) of 23.2 %.

The mean respiration rates of samples of activated sludge exposed to the test material for three hours were significantly decreased at nominal concentrations of 300 and 1 000 mg/L, with respiration rates inhibited by 36 % and 52 %, respectively. The EC20 value was statistically determined to be 107 mg/L. Owing to the upper 95 % confidence limit being out of range, there was insufficient data for an EC50 value to be determined. However as there was 52 % inhibition the EC50 was estimated to be in the order of 1000 mg/L, the highest concentration tested. The no observed effect concentration (NOEC) for the test material was 100 mg/L.

Results with reference substance (positive control):

The three-hour 50 % effect concentration (EC50) for 3,5-DCP was calculated to be 14.5 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.

Reported statistics and error estimates:

The statistical methods used in determining EC values are detailed in the statistical analysis report. The no observed effect concentration (NOEC) is the highest concentration tested that gave no statistically significant level of inhibition.

Validity criteria fulfilled:

yes

Conclusions:

The mean respiration rates of samples of activated sludge exposed to the test material for three hours were significantly decreased at nominal concentrations of 300 and 1000 mg/L, with respiration rates inhibited by 36 % and 52 %, respectively. The EC20 value was statistically determined to be 107 mg/L. Owing to the upper 95 % confidence limit being out of range, there was insufficient data for an EC50 value to be determined. However as there was 52 % inhibition the EC50 was estimated to be in the order of 1000 mg/L, the highest concentration tested. The no observed effect concentration (NOEC) for the test material was 100 mg/L.

Executive summary:

The effect of the test material on the respiration rate of activated sludge was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 209.

During the study samples of activated sludge (suspended solids 1.6 g/L), fed with synthetic sewage, were exposed to dilutions of the test material for three hours. Their rates of oxygen consumption were determined using oxygen electrodes and were compared with those of four replicate controls that contained activated sludge and synthetic sewage alone.

The nominal test material concentrations used in the study were 10, 30, 100, 300 and 1000 mg/L; four replicates were prepared for each concentration including the control. Single replicates of the reference inhibitor 3,5-dichlorophenol (3,5-DCP) were used at 3, 10 and 32 mg/L, as a positive control.

The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 22.9 mgO2/gh with a coefficient of variation (CV) of 23.2 %. The three-hour 50 % effect concentration (EC50) for 3,5-DCP was calculated to be 14.5 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.

The mean respiration rates of samples of activated sludge exposed to test material for three hours were significantly decreased at nominal concentrations of 300 and 1000 mg/L, with respiration rates inhibited by 36 % and 52 %, respectively. The EC20 value was statistically determined to be 107 mg/L. Owing to the upper 95 % confidence limit being out of range, there was insufficient data for an EC50 value to be determined. However as there was 52 % inhibition the EC50 was estimated to be in the order of 1000 mg/L, the highest concentration tested. The no observed effect concentration (NOEC) for the test material was 100 mg/L.

Description of key information

3-hour NOEC 100 mg/L, OECD 209, K Hillier

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

The effect of the test material on the respiration rate of activated sludge was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 209.

During the study samples of activated sludge (suspended solids 1.6 g/L), fed with synthetic sewage, were exposed to dilutions of the test material for three hours. Their rates of oxygen consumption were determined using oxygen electrodes and were compared with those of four replicate controls that contained activated sludge and synthetic sewage alone.

The nominal test material concentrations used in the study were 10, 30, 100, 300 and 1000 mg/L; four replicates were prepared for each concentration including the control. Single replicates of the reference inhibitor 3,5-dichlorophenol (3,5-DCP) were used at 3, 10 and 32 mg/L, as a positive control.

The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 22.9 mgO2/gh with a coefficient of variation (CV) of 23.2 %. The three-hour 50 % effect concentration (EC50) for 3,5-DCP was calculated to be 14.5 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.

The mean respiration rates of samples of activated sludge exposed to test material for three hours were significantly decreased at nominal concentrations of 300 and 1000 mg/L, with respiration rates inhibited by 36 % and 52 %, respectively. The EC20 value was statistically determined to be 107 mg/L. Owing to the upper 95 % confidence limit being out of range, there was insufficient data for an EC50 value to be determined. However as there was 52 % inhibition the EC50 was estimated to be in the order of 1000 mg/L, the highest concentration tested. The no observed effect concentration (NOEC) for the test material was 100 mg/L.