Tangled Multi-Walled Carbon Nanotubes

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

january 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
For the preliminary test, a series of 6 concentrations (100, 50, 10, 5, 1 and 0.1 mg/L) of the test item in dilution water.
Concerning the definitive test, based on the results of preliminary test, it was decided to perform a limit test at 100mg/L.

- Sampling method:
.Preliminary test:
In the preliminary test, daphnids were exposed under static conditions to a series of 6 concentrations of GRAPHISTRENGTH C100 in dilution water. The toxic effect measured during the assay was the inhibition mobility over a time period of 48 hours.
Approximately 90% of the final volume of the test suspensions was added into test flasks. Five Daphnis magna aged from 6 to 24 hours were added into each test flask, which were then totally filled with the test suspension (30 mL). Two replicates were prepared for each concentration.
.Definitive test:
Based on the results of preliminary test, it was decided to perform a lirait test at 100 mgIL. Volumetric flasks were filled with the required volumes of the test item stock suspension.
Approximately 90% of the final volume of the test suspension was added into test flasks

- Sample storage conditions before analysis:
In order to keep GRAPHISTRENGTH C100 in suspension, flasks were continuously maintained under axial rotation by use of a cylindric roller device.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

-Method:
The stock solution will be prepared by dispersing the test item in reconstituted water made up by diluting pure minerai salts (analytical grade) in ultra-pure water. The test solutions will then be prepared by diluting the stock solution in reconstituted water under stirring.

- Controls:
.Preliminary test: Two control flasks were prepared, with no test item.
.Definitive test: Four control flasks were prepared, with no test item.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):dilution water
- Concentration of vehicle in test medium (stock solution and final test solution):


- Evidence of undissolved material (e.g. precipitate, surface film, etc):no data

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain:Daphnia magna Straus
- Source:from a stock breeding in the laboratory reared in VOLVIC° water added with vitamin B12, Na2SeO3, 5H2O, calcium and magnesium salts.
Selection of test organisms less than 24 hours-old is made by filtration.
- Age at study initiation (mean and range, SD):from 6 to 24 hours
- Weight at study initiation (mean and range, SD):no data
- Length at study initiation (length definition, mean, range and SD):no data
- Valve height at study initiation, for shell deposition study (mean and range, SD):no data

- Food type: fed with unicellular green fresh-water algae Pseudokirchneriella subcapitata and Chlorella vulgaris.
- Amount:no data
- Frequency:no data

ACCLIMATION
- Acclimation period:no data

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

the end of the test (48h)

Hardness:

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

Test temperature:

continuously recorded in one test flask, remained between 18 and 22°C and was constant within ± 1°C.

pH:

measured using a METTLER TOLEDO 345 pH-meter.
Concerning pH, no information is clearly given in the document, consequently we consider that the estimation is done for relevant natural environmental conditions (pH = 5 – 9).

Dissolved oxygen:

measured using a WTW OXI 538 oxymeter.
Validity criteria: > or = 3 mg/L in control and test vessels.
measured data: at T0 and C0, Dissolved O2 = 8.4 mg/L
at T0 and C= 100mg/L, Dissolved O2 = 8.5 mg/L
at T48h and C0, Dissolved O2 = 8.1mg/L
at T48h and C= 100mg/L, Dissolved O2 = 8.3mg/L.

Salinity:

Fresh water test

Nominal and measured concentrations:

nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 30 mL glass bottles stoppered with PTFE bungs and sealed with aluminium caps. Approximately 90% of the final volume of the test suspensions was added into test flasks.

- Material, size, headspace, fill volume:
In order to keep GRAPHISTRENGTH C100 in suspension, flasks were continuously maintained under axial rotation by use of a cylindric roller device.
The appearance of the test solutions was visually checked: test item particles remains as suspension during the test.
Physical-chemical parameters were measured using a METTLER TOLEDO 345 pH-meter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement.
Each test flask was totally filled with the test suspension (30 mL).

- Aeration:until oxygen saturation.

- Type of flow-through (e.g. peristaltic or proportional diluter):

- Renewal rate of test solution (frequency/flow rate):no data
- No. of organisms per vessel:Five Daphnis magna were added into each test flask, for preliminary and definitive tests.
- No. of vessels per concentration (replicates):
Concerning the preliminary test: Two replicates were prepared for each concentration.
Concerning the definitive test: Four replicates were prepared for each concentration.
- No. of vessels per control (replicates):
Concerning the preliminary test: Two control flasks were prepared, with no test item.
Concerning the definitive test: Four control flasks were prepared with no test item.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
ultra-pure water was used for the preparation of dilution water.
Osmotically filtered water was used to prepare the dilution water:
Chemical reagents used for the preparation of dilution water are of « analytical grade ».
Dilution water is prepared according to the protocol described in standard NF EN ISO 6341:
The following stock solutions are prepared:
CaCI2, 2H2O: 11.76 g per litre of water, MERCK Ref. 1.02382.5000 batch A935182902
MgSO4, 7H2O: 4.93 g per litre of water, MERCK Ref. 1.05886.1000 batch A975086904
NaHCO3: 2.59 g per litre of water, MERCK Ref. 1.06329.0500 batch K39347429919
KCI: 0.23 g of per litre of water, MERCK Ref. 1.04936.0250 batch K37429936851

Dilution water is obtained by adding 25 mL of each of the stock solutions in a final volume of 1 litre and then aerating the solution until oxygen saturation.
Sum of Ca and Mg ions in dilution water is of 2.5 mmol/L and ratios of Ca/Mg et Na/K are 4 and 10.

- Total organic carbon:
- Particulate matter:
- Metals:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:no
- Photoperiod:no data
- Light intensity:Test flasks were incubated in darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study:
In the preliminary test, daphnids were exposed under static conditions to a series of 6 concentrations (100, 50, 10, 5, 1 and 0.1 mg/L) of GRAPHISTRENGTH C100 in dilution water.
- Test concentrations:
Based on the results of preliminary test, it was decided to perform a lirait test at 100 mgIL. Volumetric flasks were filled with the required volumes of the test item stock suspension.

Reference substance (positive control):

not required

Remarks:

Test(s) with a reference substance (Potassium dichromate) should be done preferably every month and at least twice a year.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities:no
- Observations on body length and weight:no data
- Other biological observations:no data
- Mortality of control:no
- Other adverse effects control:no
- Abnormal responses:no

Results with reference substance (positive control):

- Results with reference substance valid? yes
The sensitivity of the test system and the methodology are evaluated every month by performing an inhibition test on potassium dichromate. The nearest value of EC50 obtained on 17/02/10 was 0.78 mg/L. For reference, ISO 6341 has a validity criteria specifying that EC50 must be in the range 0.6 to 2.1 mg/L.

- Mortality:no
- EC50/LC50:0.78 mg/L

Reported statistics and error estimates:

no

Table 1: Dissolved O2 and pH measured at the beginning and at the end (48h) of the exposition.

Nominal concentration (mg/L)	pH		Dissolved O2 (mg/L)
	T0	T48h	T0	T48h
0 (T)	7.95	7.77	8.4	8.1
100	7.95	7.73	8.5	8.3

Table 2: Range finding test-Percentage of Daphnia magna immobilisation

Nominal concentration (mg/L)	Immobilisation (%)
	24h	48h
T	0	0
100	0	0
50	0	0
10	0	0
5	0	0
1	0	0
0.1	0	0

Table 3: Definitive test-Percentage of Daphnia magna immobilisation

Nominal concentration (mg/L)	immobilisation
	24h	48h
T	0	0
100	0	0

Table 4: EC50 values determined at 24 and 48h post exposure based on nominal concentrations.

Endpoint	EC50 value (mg/L)	NOEC (mg/L)
24h	>100	ND
48h	>100	ND

Validity criteria fulfilled:

yes

Remarks:

see conclusions

Conclusions:

The determination of the inhibition of mobility of Daphnia magna exposed to the test item GRAPHISTRENGTH C100 for a duration of 48 hours was assessed according to the OECD Guideline 202.
Daphnia magna were exposed under static conditions to GRAPHISTRENGTH C100 dispersed in dilution water.
The concentration of test item causing a 50 % reduction in mobility (EC50) was estimated.

Validity criteria:
- in the control (not) more that 10 per cent of the daphnids have been immobilised.
- dissolved oxygen concentration at the end of the test has (not) been greater than 3 mg/L in control and test vessels.

Executive summary:

The acute toxicity of the test item GRAPHISTRENGTH C100, was evaluated withDaphnia magna in a study performed in accordance with OECD testing guideline 202 and GLP requirements. The young daphnids were exposed to nominal test concentrations between 0.1 and 100 mg/L under static conditions during 48 hours. EC50-48 h was estimated using nominal concentrations as being greater than 100 mg/L. As no immobilisation was observed up to 100 mg/L (the highest tested concentration) no EC50 could be determined. EC0 is greater or equal to 100 mg/L.  Mortality in control, temperatures, pH range and dissolved oxygen concentration were reported and in agreement with the guideline requirements.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations:
Based on precedent results (Gancer, 2010), it was decided to only perform a limit test at 100 mg/L

- Sampling method:
Volumetric flasks were filled with the required volumes of the test item stock suspension.
Approximately 90% of the final volume of the test suspension was added into test flasks

- Sample storage conditions before analysis:
In order to keep GRAPHISTRENGTH C100 in suspension, flasks were continuously maintained under axial rotation by use of a cylindric roller device.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

-Method:
The stock solution will be prepared by dispersing the test item in reconstituted water made up by diluting pure minerai salts (analytical grade) in ultra-pure water. The test solutions will then be prepared by diluting the stock solution in reconstituted water under stirring.

- Controls:
Four control flasks were prepared, with no test item.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):dilution water
- Concentration of vehicle in test medium (stock solution and final test solution):


- Evidence of undissolved material (e.g. precipitate, surface film, etc):no data

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain:Daphnia magna Straus
- Source:from a stock breeding in the laboratory reared in VOLVIC° water added with vitamin B12, Na2SeO3, 5H2O, calcium and magnesium salts.
Selection of test organisms less than 24 hours-old is made by filtration.
- Age at study initiation (mean and range, SD):< 24 hours
- Weight at study initiation (mean and range, SD):no data
- Length at study initiation (length definition, mean, range and SD):no data
- Valve height at study initiation, for shell deposition study (mean and range, SD):no data

- Food type: fed with unicellular green fresh-water algae Pseudokirchneriella subcapitata and Chlorella vulgaris.
- Amount:no data
- Frequency:no data

ACCLIMATION
- Acclimation period:no data

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

the end of the test (48h)

Test temperature:

continuously recorded in one test flask, remained between 18 and 22°C and was constant within ± 1°C.

pH:

from 7.56 to 8.03

Dissolved oxygen:

from 7.4 to 9.0 mg O2/L

Salinity:

Fresh water test

Nominal and measured concentrations:

nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 30 mL glass bottles stoppered with PTFE bungs and sealed with aluminium caps. Approximately 90% of the final volume of the test suspensions was added into test flasks.

- Material, size, headspace, fill volume:
In order to keep GRAPHISTRENGTH C100 in suspension, flasks were continuously maintained under axial rotation by use of a cylindric roller device.
The appearance of the test solutions was visually checked: test item particles remains as suspension during the test.
Physical-chemical parameters were measured using a METTLER TOLEDO 345 pH-meter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement.
Each test flask was totally filled with the test suspension (30 mL).

- Aeration:until oxygen saturation.

- Type of flow-through (e.g. peristaltic or proportional diluter):

- Renewal rate of test solution (frequency/flow rate):no data
- No. of organisms per vessel:Five Daphnis magna were added into each test flask.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
ultra-pure water was used for the preparation of dilution water.
Osmotically filtered water was used to prepare the dilution water:
Chemical reagents used for the preparation of dilution water are of « analytical grade ».
Dilution water is prepared according to the protocol described in standard NF EN ISO 6341:
The following stock solutions are prepared:
CaCI2, 2H2O: 11.76 g per litre of water,
MgSO4, 7H2O: 4.93 g per litre of water,
NaHCO3: 2.59 g per litre of water,
KCI: 0.23 g of per litre of water,
Dilution water is obtained by adding 25 mL of each of the stock solutions in a final volume of 1 litre and then aerating the solution until oxygen saturation.
Sum of Ca and Mg ions in dilution water is of 2.5 mmol/L and ratios of Ca/Mg et Na/K are 4 and 10.

- Total organic carbon:
- Particulate matter:
- Metals:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:no
- Photoperiod:: no data
- Light intensity:Test flasks were incubated in darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Test concentrations:
Based on previous results (Gancet, 2010), it was decided to perform a limit test at 100 mgIL. Volumetric flasks were filled with the required volumes of the test item stock suspension.

Reference substance (positive control):

not required

Remarks:

Test(s) with a reference substance (Potassium dichromate) should be done preferably every month and at least twice a year.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities:no
- Observations on body length and weight:no data
- Other biological observations:no data
- Mortality of control:no
- Other adverse effects control:no
- Abnormal responses:no

Results with reference substance (positive control):

- Results with reference substance valid? yes
The sensitivity of the test system and the methodology are evaluated every month by performing an inhibition test on potassium dichromate. The nearest value of EC50 obtained on 08/08/2012 was 0.8 mg/L. For reference, ISO 6341 has a validity criteria specifying that EC50 must be in the range 0.6 to 2.1 mg/L.

- Mortality:no
- EC50/LC50:0.8 mg/L

Reported statistics and error estimates:

no

Table 1: Dissolved O2 and pH measured at the beginning and at the end (48h) of the exposition.

Nominal concentration (mg/L)	pH		Dissolved O2 (mg/L)
	T0	T48h	T0	T48h
0 (T)	8.02	7.56	9.0	7.4
100	8.03	7.96	9.0	8.2

Table 2: Definitive test-Percentage of Daphnia magna immobilisation

Nominal concentration (mg/L)	immobilisation
	24h	48h
T	0	0
100	0	0

Table 3: EC50 values determined at 24 and 48h post exposure based on nominal concentrations.

Endpoint	EC50 value (mg/L)	NOEC (mg/L)
24h	>100	ND
48h	>100	ND

Validity criteria fulfilled:

yes

Remarks:

see conclusions

Conclusions:

The determination of the inhibition of mobility of Daphnia magna exposed to the test item GRAPHISTRENGTH C100 for a duration of 48 hours was assessed according to the OECD Guideline 202.
Daphnia magna were exposed under static conditions to GRAPHISTRENGTH C100 dispersed in dilution water.
The concentration of test item causing a 50 % reduction in mobility (EC50) was estimated to be >100 mg/L (nominal concentration).

Validity criteria:
- in the control (not) more that 10 per cent of the daphnids have been immobilised.
- in the control no daphnids have been trapped at the surface of the water
- dissolved oxygen concentration at the end of the test has (not) been greater than 3 mg/L in control and test vessels.

Executive summary:

The acute toxicity of the test item GRAPHISTRENGTH C100, was evaluated withDaphnia magna in a study performed in accordance with OECD testing guideline 202 and GLP requirements. The young daphnids were exposed to a nominal test concentration of 100 mg/L under static conditions during 48 hours. EC50-48 h was estimated using nominal concentrations as being greater than 100 mg/L. As no immobilisation was observed up to 100 mg/L no EC50 could be determined. EC0 is greater or equal to 100 mg/L.  Mortality in control, temperatures, pH range and dissolved oxygen concentration were reported and in agreement with the guideline requirements.

Description of key information

The acute toxicity of GRAPHISTRENGTH C100 MWCNTs on Daphnia magna was evaluated twice in accordance with OECD TG 202 and GLP requirements.
In the study from Gancet(2010), daphnids were exposed to nominal test concentrations between 0.1 and 100 mg/L under static conditions during 48 hours.As no immobilisation was observed up to 100 mg/L (the highest tested concentration) no EC50 could be determined. EC0 is greater or equal to 100 mg/L

This result was confirmed by a second study performed by Gancet C. in 2012.