Single Wall Carbon Nanotubes (SWCNT)

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Sep - 5 Oct 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

943-098-9

Cas Number:

7440-44-0

Molecular formula:

Carbon. Morphological features: - Tube cylindrical surface is formed by 6-membered rings consisting of carbon atoms linked by a double bond. - Carbon nanotube chirality is random. - Tube length: 1 - 10 μm. - Agglomeration: CNTs tend for form bundles

IUPAC Name:

carbon

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Remarks:

Tween 80

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
1 mg/L stock solution was made by crushing freeze dried sample and dispersing with Tween 80 (10 mg/L test water).
Preparation of vehicle and test item stock solution in detail:
First a stock solution (A) with the vehicle Tween 80 was prepared by adding 10 mg Tween 80 to 1 L ultrapure water (10 mg/L) under stirring. Stock solution (A) was kept refrigerated in a dark place until further use. A second stock solution (B) with a Tween 80 concentration of 1 mg/L was prepared by adding stock solution (A) to test medium. Also stock solution (B) was kept refrigerated in a dark place until further use.
A first test item stock solution (C) was prepared in a glass vessel by adding 50 mg test item to 50 mL stock solution (A). Those were mixed with a stirrer while additionally being sonicated at the same time. Inversion stirring was applied as needed up to 6 h until a good dispersion of test material was achieved. Concentration of stock solution (C) was 1 mg test item/L and 10 mg Tween 80/L. The stock solution (C) was stored at room temperature and showed to be stable for 21 d.
A second test item stock solution (D) was prepared, achieving a nominal test item concentration of 100 mg/L and and Tween 80 concentration of 1 mg/L. Therefore, a respective amount of stock solution (C), which was previously sonicated for 10 min, was added to test medium in a measuring bottle. As mixing method sonication took place for 10 min. Stock solution (D) was directly used and not further stored.
The actual test solution was prepared in a measuring flask by mixing a respective amount of stock solution (B) and (D) with test medium using inversion stirring. Therefore, adequate volumes of stock solutions (B and D) were taken while keeping the stock solution (D) being stirred.
- Controls: Water, vehicle
- Chemical name of vehicle: Tween 80

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea / Daphnia magna
- Age at study initiation: < 24 h
- Source: National Institute for Environmental Studies, Ministry of the Environment
- Age of parental stock: 3 - 4 weeks
- Feeding during test: Yes
- Food type: Chlorella vulgaris
- Amount: 0.15 mg C (organic carbon content)/daphnia/day
- Frequency: Daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Animals were kept in 2L Elendt M4 medium and groups of ≤ 25 (20±1°C; pH 6 - 9; dissolved oxygen ≥ 60 %; Light conditions: 16 h light (800 lux) : 8 h darkness). First renewal of medium took place within the first 5 days of starting the culture, subsequently a change was made 3 times per week. Animals were kept for max 28 days, juveniles were removed daily.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

<250 mg CaCO3/L

Test temperature:

20 ± 1 ℃

pH:

6 - 9 ( variation < 1.5)

Dissolved oxygen:

≥ 3 mg/L (no aeration)

Nominal and measured concentrations:

Nominal: 0 (control), 0 (vehicle control), 0.01, 0.032, 0.10, 0.32, 1.0 mg test substance/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beaker
- Type: Closed (loosely with a PVC lid)
- Material, size, headspace, fill volume: Glass, 100 mL, with headspace, 80 mL
- Aeration: No aeration during the test. Medium was thoroughly aerated before usage in the test.
- Renewal rate of test solution: every 2 days
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10
- No. of vessels per vehicle control: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium (water was prepared as described in OECD Guidelines for Testing of Chemicals 211)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light – 8 h dark
- Light intensity: ≤800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Date of first offspring
- Number of living offspring, check for the presence of dead offspring, aborted eggs, or ephippial eggs (daily, starting from day 7 of the test on)
- Mortality, mobilization status and abnormal appearance of the parent animals (all parameters were observed daily)
- For reference data, the living parent Daphnia in each test group were examined microscopically at the end of exposure (non-GLP procedure).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ≤ 3.2
- Range finding study: Yes, semi-static acute range finding tests with D.magna according to TG OECD 202 were carried out to gain information about the vehicle (Tween 80) and highest test concentration to be used within the main test.
- Test concentrations: Control and vehicle control, 0.0030, 0.03, 0.3 and in a 10 mg test item/L
- Results used to determine the conditions for the definitive study: The choice of concentrations for the main test based on the 48 h acute semi-static range finding tests.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used. The sensitivity of Daphnia was investigated periodically (about every 6 months) by conducting an acute immobilization test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.62 mg/L

95% CI:

>= 0.42 - <= 1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Could not be determined by statistical calculation because the mortality of parent Daphnia at the maximum concentration level was less than 50 %. Therefore, the LC50 value is provided as an estimated concentration range.

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Other biological observations: In the highest concentration, all 10 parent Daphnia were smaller and whiter in appearance than in the vehicle control after day 6 of exposure.
Date to first offspring: In the control and the auxiliary agent control, the first brood day of the parent Daphnia was within 8 days of the start of exposure. In concentrations 1 - 5, the first brood day was within 9 days of the start of exposure.
- Mortality of control: 10 % (control) 0 % ( vehicle control)
- Effect concentrations exceeding solubility of substance in test medium: Yes, substance is insoluble

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 48 h EC50 for potassium dichromate = 0.71 mg/L (95 % CI: 0.62 - 0.82 mg/L)
- Other:
Results from June 1998 onwards:
Mean ± standard deviation: 48 h EC50 for potassium dichromate: 0.74 ± 0.12 mg/L, n=27
(min. value–max. value: 0.57–1.02 mg/L)

Reported statistics and error estimates:

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were evaluated by ANOVA and Dunnett's multicomparison test) using the mean number of living offspring (while not taking into account the mortality of the parent Daphnia).

Any other information on results incl. tables

Table 1: Mean Cumulative Number of Offsprings Produced per Parent Alive (ΣF1/P)

Test Group	Nominal Conc. (mg/L)	ΣF1/P
		Exposure time (days)
		7.00	8.00	9.00	10.00	11.00	12.00	13.00	14.00	15.00	16.00	17.00	18.00	19.00	20.00	21.00
Control	--	0.00	17.90	17.90	17.90	17.90	56.60	56.60	87.00	87.00	87.00	87.00	102.30	102.30	102.30	103.40
Solv. control	--	7.60	15.70	15.70	44.20	44.20	54.20	67.50	100.00	100.00	111.30	111.30	130.90	134.30	148.00	160.90
1	0.010	5.40	14.90	14.90	35.30	35.30	58.00	71.40	106.90	106.90	114.70	114.70	131.80	131.80	149.90	160.90
2	0.032	9.80	14.90	14.90	44.30	44.30	55.00	84.10	104.10	104.10	124.80	124.80	135.90	139.40	158.70	168.00
3	0.10	11.00	16.80	16.80	49.30	49.30	56.60	91.20	103.50	103.50	131.10	131.10	138.80	143.70	172.00	172.00
4	0.32	8.30	11.70	11.70	46.00	46.00	48.70	86.10	90.90	90.90	125.60	125.60	128.00	138.60	159.60	161.20
5	1.00	0.00	13.50	13.50	13.50	13.50	35.50	35.50	54.00	54.00	54.00	54.00	82.00	82.00	102.00	102.00

 

Table 2: Reproduction Rate, Inhibition Rate and Results of Statistical Comparison

Test Group	Nominal Conc. (mg/L)	Cumulative Number of Offsprings										Mean	S.D.	Reprod. Rate (%)	Inhibit.Rate	Signific.diff.
		Vessel No.
		1	2	3	4	5	6	7	8	9	10
Control	--	133	137	135	D	79	138	130	138	141	143	130.4	19.7	--	--	--
Solvent control	--	125	146	145	149	180	163	171	184	179	167	160.9	19.1	--	--	--
1	0.010	154	139	166	193	150	D	174	149	161	162	160.9	15.9	100	0	-
2	0.032	137	156	165	183	189	138	187	171	D	186	168	20.5	104.4	-4.4	-
3	0.10	179	173	183	159	150	187	141	186	188	174	172	16.6	106.9	-6.9	-
4	0.32	106	D	167	161	177	167	169	173	164	167	161.2	21.2	100.2	-0.2	-
5	1.00	102	D	D	D	D	D	D	D	102	D	102	--	63.4	36.6	+

Equation B = A/A solvent control > 100
C = 100-B
D: Not included for calculation because the parental Daphnia was dead during a 21-day exposure period.
- : Indicates no significant difference from the solvent control
*: Indicates a significant difference (α=0.05) from the solvent control (there was no sign. in this test)
**: Indicates a significant difference (α=0.01) from the solvent control (there was no sign. in this test.)
+: Statistical analysis were performed except Conc. 5 which was 80 % of cumulative mortality of parent Daphnia. S significant
There is no difference (α=0.05) between the concentration groups except Conc. 5 and the solvent control

 

Table 3: See attached background material "Table 3" (Content: Cumulative mortality of parent Daphnia over 21 d exposure to the test item)

 

Table 4: See attached background material "Table 4" (Content: Observations for Daphnia during the 21 d reproduction test including detailed offspring, moratility and behaviour data)

 

Table 5: Validity criteria for OECD 211

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.	10 %, (0 % vehicle controll)	Yes
The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60 in the control.	≥ 79	Yes

 

Pictures 1-7: See attached background material "Pictures 1-7" (Content: Microscopic observations of Daphnia after 21 d exposure [non-GLP])

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Please refer to Table 5 under 'Any other information on results incl. tables' for more details

Conclusions:

The long-term test with D. magna revealed a 21-d NOEC of 0.32 mg/L based on reproduction and nominal test item concentrations.