Tangled Multi-Walled Carbon Nanotubes

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August-October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

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):
no data

Analytical monitoring:

no

Details on sampling:

- Concentrations:
.In the preliminary test: 100, 50, 10, 5 and 1 mg/L of GRAPHISTRENGTH C100 in dilution water.
.In the definitive test: 100, 61, 37, 22, 13.5, 8 and 5 mg/L.

- Sampling method:
.For preliminary test:
suspensions of GRAPHISTRENGTH C100 were directly prepared for each concentration by weighing 1, 5, 10, 50 and 100 mg into 100 mL of dilution water and adjusted to 1L.
Then 50 mL of each suspension under stirring were transfered to the corresponding test flasks, where algae were added to reach 10^4 cells per mL.
After 24, 48 and 72h of incubation, a volume of 200µL was sampled from each test flask, pipetted into a quartz microplate.
Inoculated control flask (T) and non-inoculated blank (BI) were prepared and incubated.

.For definitive test:
suspensions of GRAPHISTRENGTH C100 were directly prepared for each concentration by weighing 10, 22, 48, 105, 230, 500 and 1000 mg into 100 mL of dilution water and adjusted to 1L.
Then 50 mL of each suspension under stirring were transfered to the corresponding test flasks, where algae were added to reach 10^4 cells per mL.
After 24, 48 and 72h of incubation, about 1 mL was sampled from each test flask under.
Inoculated control flask (T) and non-inoculated blank (BI) were prepared and incubated.

- Sample storage conditions before analysis:samples were stored in darkness until determination of algae concentration.

Vehicle:

no

Details on test solutions:

Suspensions of Test item were prepared by weighing the required quantity in 30 mL of dilution water and adjusting to 1 L. Then, 50 mL of each suspenson under stirring were transferred to the test flasks, and algae were added such that reaching 10E4 cells/mL

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM

- Strain:Pseudokirchneriella subcapitata, CCAP 278/4 stock
- Source (laboratory, culture collection):the Culture centre of Algae and Protozoa
- Age of inoculum (at test initiation):no data

- Method of cultivation: described in Annex 2 of the OECD 201 guideline.
Two flaks, each containing approximately 100 mL of axenic stock culture of algae are incubated at 23+/-1°C under ligthing, slowly continuously shaken. these stock cultures are renewed every week, using two new cultures.
The quality of the stock culture was verified for the absence of micro-organisms and deformed cells under microscopic observation before use.
Three days before the beginning of the study, two pre-cultures were prepared by inoculating each stock suspension of algae (5 mL) into sterile dilution water (500 mL). the pre-cultures were incubated under the same conditions as those used for the stock cultures. Only one of the two pre-cultures was used to inoculate the test flasks. the second one was to be used only if the first one was damaged.



ACCLIMATION
- Acclimation period:no data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

According to annex 3 of OECD TG 201

Test temperature:

23+/-1°C

pH:

measured using a METTLER TOLEDO 345.
Nominal Concentration pH
mg/L T0 T72h
0 (Bl) 7.97 8.03
0 (T) - 9.79
100 7.97 8.03
61 7.97 8.02
37 7.97 8.05
22 7.97 8.20
13.5 7.97 8.61
8 7.97 8.90
5 7.98 9.09

Dissolved oxygen:

measured using a WTW OXI 538 oxymeter.

Nominal Concentration Dissolved O2 (mg/L)
mg/L T0 T72h
0 (Bl) 9.4 8.7
0 (T) - 9.7
100 9.2 8.7
61 9.2 8.7
37 9.1 8.7
22 9.1 9.0
13.5 9.3 9.2
8 9.2 9.3
5 9.1 9.4

Salinity:

fresh water

Nominal and measured concentrations:

nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 mL glass bottles stoppered with cellulose bungs.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:
The incubation was performed in a phytoculture cabinet Strader DCS Pulsar.
Flasks carrying trays are continuously rotating at 20 rpm and constantly illuminated.
It was observed that most of GRAPHISTRENGTH C100 particles did not remain as suspension and gathered together in the lower part of each flask.
Filling volume: 50 mL.
Determination of algae concentration was carried out using cytofluorimetry with a Cytofluor 2350 device.

- Aeration: passive
- Initial cells density:10^4 cells/mL

- No. of vessels per concentration (replicates): concerning the definitive test, test flasks and blanks were prepared in triplicate.
- No. of vessels per control (replicates):6 replicates were used for the control.

GROWTH MEDIUM
- Standard medium used: according to annex 2 of OECD TG 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the protocol described in the Annex 3 of the OECD Guideline 201.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 hour illumination
- Light intensity and quality:flasks are illuminated by 8 fluorescent tubes between 6,000 and 10,000 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cytofluorimeter with a Cytofluor 2350 device.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI = 12 - 29

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI = 3.4 - 12

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% CI = 4.1 - 10

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% CI = 0.65 - 3.5

Details on results:

Dissolved oxygen and pH were measured in the test solutions at the beginning and at the end of the test. Measurements were carried out in blank non-inoculated solution at T0 and T72 and in control inoculated solution at T72.

It was observed that most of GRAPHISTRENGTH C100 particles did not remain as suspension and gathered together in the lower part of each flask.
Microscopic observation confirmed that the algae appeared normal at the end of the test.

An increase in the pH is observed. This may be associated to consumption of the dissolved CO2 due to the growth of algae.

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

The growth inhibition data are analyzed using an Excel program. It was designed to calculate the EC50 value and the 95% confidence interval. Probit analysis is generally used to calculate the 24, 48 and 72-hour ECx values.

Nominal concentration	Average cell density (cell/mL)
mg/L	T0	T24h	T48h	T72h
T	1.00E+04	2.79E+04	9.86E+05	6.67E+06	1.04E+08	2.168
100	1.00E+04	1.75E+03	5.59E+04	0.00E+00	7.83E+05	0.000
61	1.00E+04	1.75E+03	9.95E+04	8.73E+03	1.94E+06	-0.045
37	1.00E+04	1.05E+04	9.95E+04	3.67E+04	2.48E+06	0.433
22	1.00E+04	2.10E+04	2.41E+05	1.03E+06	1.81E+07	1.546
13.5	1.00E+04	1.40E+04	4.00E+05	2.24E+06	3.62E+07	1.804
8	1.00E+04	3.14E+04	6.41E+05	3.39E+06	5.63E+07	1.942
5	1.00E+04	2.62E+04	6.91E+05	3.42E+06	5.77E+07	1.945

Nominal concentration	IAi	Iµi
(mg/L)	(%)	(%)
T	0.00	0.00
100	99.25	100.00
61	98.14	102.09
37	97.61	80.02
22	82.59	28.70
13.5	65.09	16.77
8	45.80	10.39
5	44.45	10.27

 

Validity criteria fulfilled:

yes

Conclusions:

Growth inhibition of a culture of freshwater algae Pseudokirchneriella subcapitata by MWCNTs was observed.

Validity criteria:
The biomass in the control cultures increased exponentially by a factor of 667 (corresponding to a specific growth rate of 2.168 day-1) within the 72-hour test period, which is higher than the minimal value (R=16) mentioned OECG Guideline 201.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35%. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Executive summary:

The determination of the growth inhibition of the fresh water algae Pseudokirchneriella subcapitata exposed to the test item GRAPHISTRENGTH C100 for a duration fo 72 hours was assessed according to the OECD Guideline 201.

Algae were exposed to a range of concentrations of GRAPHISTRENGTH C100 dispersed in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.

The concentrations of the test item causing 50% reduction in growth rate (ErC50) and in biomass (EbC50) were estimated. EC10were also calculated.

ErC50-72h, based on growth rate, was 19 mg/L. EbC50-72h, based on biomass, was of 7.2 mg/L

ErC10-72h, based on growth rate, was 7.8 mg/L. EbC10-72h, based on biomass, was of 1.9 mg/L

Description of key information

Growth inhibition of freshwater unicellular green alga P. subcapitata has been observed when exposed to Graphistrength C100 MWCNTs at nominal concentrations above 10 mg/L. Investigation about causes of this inhibition argue in favour of a depletion of the growth factor iron in the medium (through adsorption to the CNTs presumably).

Key value for chemical safety assessment

EC50 for freshwater algae:

19 mg/L

EC10 or NOEC for freshwater algae:

7.8 mg/L

Additional information

Three OECD 201 studies have been carried out on Graphistrength C100 MWCNTs differing by conditions of synthesis such as the type of catalyst used and corresponding remaining as impurities in the end product. The two older studies (2010) were carried out on R&D samples, when the last one was carried out on a sample representing the industrial pilot production. The later will be consequently used as the key study.

They all show similar inhibition of growth on Pseudokirchneriella subcaptitata. Further investigations were carried out further to the 2 first studies in order to identify causes for the slight inhibition of cell growth observed. It has been shown (internal report Arkema GRL ref.: SID 15332) that standard algae test medium stirred with Graphistrength C100 at a ratio of 1 g/L is depleted in iron: instead of a nominal concentration of 16.5 mg/L, iron concentration is reduced to < 1 mg/L (LOQ). This is interpreted as a possible cause for the lack of growth in the OECD 201 experiments.