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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-19 to 2016-12-25
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 41251
- Expiration date of the lot/batch: 2017-03-01 (nominal expiry date) (taken from label)
- Purity test date: 2016-12-20 2016-12-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable under storage conditions until 01 March 2017 (nominal expiry date) (taken from label)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless liquid

OTHER SPECIFICS:
Molecular weight: 320
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Vapour Pressure: 121 mmHg at 25°C
Specific gravity/density: 1.67 at 25°C
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.10, 1.0, 10 and 100% of a saturated solution (SS) at 100 mg/L
- Sampling method: The batch of AsahiklinTM AC-2000 tested was a colourless liquid with a purity of 100.00% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying one day of slow magnetic stirring in a closed vessel to reach the maximum dissolution of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one hour where after the clear and colourless saturated solution (SS) was siphoned out and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Sample storage conditions before analysis: At room temperature
Vehicle:
yes
Remarks:
Adjusted ISO medium
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions - The batch of AsahiklinTM AC-2000 tested was a colourless liquid with a purity of 100.00% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions started with a loading rate of 100 mg/L applying one day of slow magnetic stirring in a closed vessel to reach the maximum dissolution of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one hour where after the clear and colourless saturated solution (SS) was siphoned out and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: Test medium without test item or other additives
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Justification for species other than prescribed by test guideline: This system has been selected as an internationally accepted invertebrate species
- Source: In-house laboratory culture with a known history
- Age of parental stock (mean and range, SD): For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions (same as test or not): not specified
- Type and amount of food: a suspension of fresh water algae
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:

Breeding
Start of each batch With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 8 8.8 mg/L
NaHCO3 4 6.7 mg/L
KCl 4.2 mg/L

Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reachthe following concentrations:

Trace elements: B 0.125 mg/L

Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rband Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L

Macro nutrients: Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L

Vitamins: Thiamine 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L

The hardness: 180 mg/Lexpressed as CaCO3 and the pH: 7.7 ± 0.3.
Test type:
static
Water media type:
other: Adjusted ISO medium
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not specified
Test temperature:
19 - 20°C
pH:
Start (t = 0 h) = 7.9
End (t = 48 h) = 7.8
Dissolved oxygen:
Control:
Start (t = 0 h) = 9.2
End (t = 48 h) = 9.0

Test Material
Start (t = 0 h) = 9.0
End (t = 48 h) = 8.7
Nominal and measured concentrations:
Analysis of the samples taken from 10 and 100% of the SS at the start of the test showed measured concentrations of 0.089 and 1.1 mg/L, respectively. Both concentrations remained stable during the 48-hour test period and were 91-119% of the respective initial concentrations at the end of the test. Measured concentrations in the samples taken from 0.10 and 1.0% of the SS were at or below 0.008 mg/L, which was the concentration of the lowest calibration solution.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Type (delete if not applicable): closed airtight without headspace
- Material, size, headspace, fill volume: Glass without Headspace
- Aeration: No aeration of the test solutions.
- Renewal rate of test solution (frequency/flow rate): Not specified
- No. of organisms per vessel: 5 per vessel containing 60 mL of test solution
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Please see "Composition of medium M7" in the Section 'Details on test organisms above'

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: 16 hours photoperiod daily
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for the control and the highest test concentration.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: A combined limit/range-finding test was performed, exposing twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to 100% of the SS. In addition ten daphnids per group (two replicates, five daphnids per replicate) were exposed to 0.10, 1.0 and 10% of the SS. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.
- Results used to determine the conditions for the definitive study: Analysis of the samples taken from 10 and 100% of the SS at the start of the test showed measured concentrations of 0.089 and 1.1 mg/L, respectively. Both concentrations remained stable during the 48-hour test period and were 91-119% of the respective initial concentrations at the end of the test. Measured concentrations in the samples taken from 0.10 and 1.0% of the SS were at or below 0.008 mg/L, which was the concentration of the lowest calibration solution. Given these analytical results, the EC50 was based on the measured initial concentration of the 100% SS.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K44879664) was obtained from Merck, Darmstadt, Germany.
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Measured concentrations:
Analysis of the samples taken from 10 and 100% of the SS at the start of the test showed measured concentrations of 0.089 and 1.1 mg/L, respectively. Both concentrations remained stable during the 48-hour test period and were 91-119% of the respective initial concentrations at the end of the test. Measured concentrations in the samples taken from 0.10 and 1.0% of the SS were at or below 0.008 mg/L, which was the concentration of the lowest calibration solution. Hence, the EC50 was based on the measured initial concentration of the 100% SS.

Immobility:
After 48 hours of exposure no (significant) immobility was observed at any of the test concentrations and the control. Note that a maximum of 10% immobility is accepted for the control.

Determination of effect concentrations:
The 24-hour and 48-hour EC50 for the test material was determnined to be >1.1 mg/L.

Experimental conditions:
Test conditions (pH and oxygen concentrations (mg/L)) remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: ≥3 mg/Lat the end of the test). The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).
Results with reference substance (positive control):
- Results with reference substance valid?: Yes
- Relevant effect levels: The actual responses in this reference test with K2Cr2O7 were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was within the expected range of 0.28 to 0.9 mg/L. Hence, the sensitivity of the daphnia was within the range
determined with the historical data collected at Charles River Den Bosch. The 24h-EC50 was 0.91 mg/L with a 95% confidence interval ranging from 0.77 to 1.1 mg/L. The 48h-EC50 was 0.58 mg/L with a 95% confidence interval ranging from 0.49 to 0.67 mg/L.

Table 1. Number of introduced daphnids and incidence of immobility

 

Time (h)

 

Replicate

 

Control

(<0.008)

AsahiklinTMAC-2000 (mg/L), %SS at 100 mg/L

0.10

(<0.008)

1.0

(<0.008)

10

(0.089)

100

(1.1)

 

 

0

A

5

5

5

5

5

B

5

5

5

5

5

C

5

 

 

 

5

D

5

 

 

 

5

Total

Introduced

20

10

10

10

20

 

 

 

 

24

A

0

0

0

0

0

B

0

0

0

0

0

C

0

 

 

 

0

D

0

 

 

 

0

Total

Immobilized

0

0

0

0

0

Effect %

0

0

0

0

0

 

 

 

 

48

A

0

0

0

0

1

B

0

0

0

0

0

C

0

 

 

 

0

D

0

 

 

 

0

Total

Immobilized

0

0

0

0

1

Effect %

0

0

0

0

5

 

Table 2. Effect parameters

Parameter

AsahiklinTMAC-2000 initial concentration

(mg/L)

24-h EC50

>1.1

48-h EC50

>1.1

 

Table 3. pH and oxygen concentrations (mg/L)

AsahiklinTMAC-2000 initial concentration

(mg/L)

Start (t=0 h)

End (t=48 h)

pH

O2

pH

O2

Control

7.9

9.2

7.8

9.0

1.1

7.9

9.0

7.8

8.7

Validity criteria fulfilled:
yes
Conclusions:
The 48h-EC50 was >1.1 mg/L based on a measured initial and stable concentration.
Executive summary:

The acute toxicity potential of AsahiklinTM AC-2000 was evaluated in Daphnia magna in a key guideline (OECD 202) study.

 

The batch of AsahiklinTM AC-2000 tested was a colourless liquid with a purity of 100.00% and not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying one day of slow magnetic stirring in a closed vessel to reach the maximum dissolution of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one hour where after the clear and colourless saturated solution (SS) was siphoned out and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

 

A combined limit/range-finding test was performed, exposing twenty daphnids per group (four replicates, five daphnids per replicate) to a control and to 100% of the SS. In addition, ten daphnids per group (two replicates, five daphnids per replicate) were exposed to 0.10, 1.0 and 10% of the SS. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

Analysis of the samples taken from 10 and 100% of the SS at the start of the test showed measured concentrations of 0.089 and 1.1 mg/L, respectively. Both concentrations remained stable during the 48-hour test period and were 91-119% of the respective initial concentrations at the end of the test. Measured concentrations in the samples taken from 0.10 and 1.0% of the SS were at or below 0.008 mg/L, which was the concentration of the lowest calibration solution. Given these analytical results, the EC50 was based on the measured initial concentration of the 100% SS. The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The 48h-EC50 was determined to be >1.1 mg/L, based on a measured initial and stable concentration.

Description of key information

The 24-h and 48h- EC501,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane in Daphnia magna was determined to be >1.1 mg/L, based on a measured initial and stable concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.1 mg/L

Additional information

In a key Guideline (OECD 202) short-term toxicity study in aquatic invertebrates (Charles River Laboratories Den Bosch BV., 2017b), Daphnia magna was exposed to the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane dissolved in an adjusted ISO medium) at concentrations of 0.10, 1.0, 10, and 100% of a saturated solution (SS) at 100 mg/L for a period of 48 hours.

 

The 24-h and 48h- EC501,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane in Daphnia magna was determined to be >1.1 mg/L, based on a measured initial and stable concentration.

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