Tangled Multi-Walled Carbon Nanotubes

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:

April 2010

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Stability in water: insoluble according to the SDS

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Limit test:
As precedent toxicity experiments showed very low effect, it was decided to test GRAPHISTRENGTH C 100 at final concentrations of 500 mg/L and 5000 mg/L.
Each suspension was prepared separetely by weighing of the convenient amount of the test item in 16 mL of the synthetic sewage feed and dilution to the final concentration.

Implementation of tests:
The tests were carried out in 500 ml flasks in the following repartition:
16 mL of the synthetic sewage feed containing the test item, adequate volume of ultra-pure water, 20 mL of sludge suspension.
At T0, the first control (control 1) without test item is prepared.
Then, every 15 min, the other vessels with different concentrations of item are prepared in turn.
Finally the second control (control 2) is prepared.
All suspensions are aerated and stirred during 3 hours and incubated at 20+/-2°C without light.
Then a sample of each mixture is poured into a BOD bottle.
The oxygen electrode is inserted into the bottle. The oxygen concentration is measured every minute in a period of 10 min.
The dissolved oxygen concentrations are determined electrochemically using an oxygen electrode and meter.

- Sampling method:
Test solution of 3-5 dichlorophenol:
A 0.5 g/L stock solution of 3-5 dichlorophenol in ultra-pure water is used for preparing the different tested solutions from 2.5 to 40 mg/L with factor of 2.5.

- Composition of synthetic sewage feed:
A synthetic sewage feed is made by dissolving the following solutions in 1 L of ultra-pure water:
16 g peptone, 11 g meat extract, 3 g urea, 0.7 g sodium chlorure, 0.4 g calcium chlorid dihydrate CaCl3, 2H2O
0.25 g magnesium sulfate heptahydrate Mg SO4, 7 H2O
2.8 g di-potassium hydrogen phosphate K2HPO4.

Vehicle:

yes

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Preparation of inoculum:
The inoculum comes from the activated sludge of the biological domestic wastewater treatment plant from the city of Abidos.
The effluent, obtained one day before seeding the flasks, is centrifuged at approximately 20 °C for 20 min at 4000g.
The base is re-suspended in ultra-pure water in order to keep the concentrations unchanged with respect to the sample.
The amount of dry material in the inoculum is determined by dessiccation at 105°C until a constant weight
From this result the amount of wet sludge can be calculated in order to obtain an activated sludge with a micture liquor suspended solids level of 4 g/L. Teh inoculum is preconditioned (aeration). Every day, 50 ml/L of synthetic sewage feed are added until the day the flasks are inoculated.
Values of suspended solids was 4.6 g/L. Then a dilution was ued to obtain 1 g/L. This level corresponds to a dose of 0.4 g/L in the test medium.

Test type:

other: stirring by means of a covered magnet

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

observation after the 3 h of exposure

Hardness:

Concerning hardness, no information is clearly given inthe document, consequently we consider that the estimation is done for 10-250 mg CaCO3/L.

Test temperature:

20+/2°C

pH:

Concerning pH, no information is clearly given in the document, consequently we consider that the estimation is done for 6.0-8.5 .

Dissolved oxygen:

Concerning dissolved oxygen, no information is clearly given inthe document, consequently we consider that the estimation is done for at least 60% of the air saturation.

Salinity:

Fresh water test

Nominal and measured concentrations:

2.5, 6.4, 16 and 40 mg/L for the principal tests
500 and 5000 mg/L for the limit test

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 5 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC0

Effect conc.:

>= 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

The sensitivity of the test system and the methodology is evaluated with 3,5 dichlorophenol.
An EC50-3h value of 14 mg/L was obtained, which is within the validity specified range from 5 to 30 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Quality criteria of the study:
- The difference in respiration rates between the two controls was 5% which is below 15%
- the EC50 of 3,5 dichlorophenol is 14 mg/L which is between 5 and 30 mg/L

Executive summary:

The purpose of the study was to determine the effect of the test item on bacteria according to the OECD guideline 209 “Activated sludge, respiration inhibition test”

The effect is assessed by measuring the microorganisms respiration rate on a sample of activated sludge during an incubation period of 3 hours.

The method was applied with respect to the different criteria:

        - The difference of respiration rates between the two controls was below 15%

        - EC50 of the control test with the reference item 3,5 dichlorophenol was between 5 and 30 mg/L (14 mg/L)

Two nominal concentrations were tested: 500 and 5000 mg/L. GRAPHISTRENGTH C 100 did not show any effect on the respiration of the activated sludge in the conditions of the test at the concentration of 500 mg/L. At 5000 mg/L an inhibition of respiration of 42.6 % was observed. In conclusion, EC50-3h is higher than 5000 mg/L

Description of key information

GRAPHISTRENGTH C 100 did not show any effect on the respiration of the activated sludge in the conditions of the OECD 209 test at the concentration of 500 mg/L. At 5000 mg/L an inhibition of respiration of 42.6 % was observed. In conclusion, EC50-3h is higher than 5000 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

500 mg/L