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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1995-05-10 to 1995-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study performed under GLP with no deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: FIFRA Guideline No. 72-2
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): β-carotin
- Physical state: brownish red, fine crystaline powder with clumps
- Analytical purity: 99.8%
- Purity test date: 13.07.94
- Lot/batch No.: 405108
- Expiration date of the lot/batch: 27.10.95
- Stability under test conditions: stable at room temperature
- Storage condition of test material: to be kept dry and dark at room temperature, protected from oxygen

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10, 18, 32, 58, 100 mg/L (nominal)
- Sampling method: Water samples of each concentration were taken at 0 and after 48 hour exposure and analysed for content of the test item.


Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200.2 mg test substance were added to 200 mg Tween 80/dimethylformamide (4:96, v/v) and 200 mL reconstituted water (M4 medium) and mixed for 5 minutes with a Polytron-mixer. This dilution was filled up to 2000 mL with M4-medium.
- Differential loading: No
- Controls: Blank vehicle solution, water control (M4-medium)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tween 80/dimethylformamide (4:96, v/v)
- Concentration of vehicle in test medium (stock solution and final test solution): 94.7, 86.3, 71.6, 44.2 and 0 µL
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test item precipitated to a high degree when diluted with test medium.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia Magna STRAUS
- Strain: Clon 5
- Source: in-house cultures
- Age at study initiation: <24hr
- Feeding during test: No
- Feeding during culturing: yes
- Food type: suspension of green algae (Scenedesmus subpicatus)
- Amount: density of 1.0 mL/L of Scenedesmus subspicatus suspension, density of approx. 10^8 cells/mL
- Frequency: daily (with the exception of the weekend)


ACCLIMATION
No acclimation. However, about 24 hours before test initiation young daphnids were separated from reproductive specimens by sieving all individuals through a 1500 µm sieve. This procedure was repeated before the start of exposure. The young daphnids received were retained for the test.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
257 mg CaCO3/L
Test temperature:
Start of test: 21 °C
End of test: 23 °C
pH:
Start of test: 8.0 - 8.2
End of test: 7.9 - 8.1
Dissolved oxygen:
Start of test: 95 - 100 % O2
End of test: 93 - 99 % O2
Nominal and measured concentrations:
- Nominal: 10, 18, 32, 58 and 100 mg/L
- Measured, start of test:
Nominal 10 mg/L: 1.0 mg/L, <1 mg/L
Nominal 18 mg/L: 1.5 mg/L, 1.0 mg/L
Nominal 32 mg/L: 2.7 mg/L, 1.9 mg/L
Nominal 58 mg/L: 4.5 mg/L, 3.3 mg/L
Nominal 100 mg/L: 6.2 mg/L, 4.5 mg/L
- Measured, end of test:
Nominal 10 mg/L: <1.0 mg/L, <1 mg/L
Nominal 18 mg/L: <1.0 mg/L, <1 mg/L
Nominal 32 mg/L: <1.0 mg/L, <1 mg/L
Nominal 58 mg/L: <1.0 mg/L, <1 mg/L
Nominal 100 mg/L: 1.8 mg/L, 2.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass beakers containing 100 mL of test medium, the blank vehicle or the blank M4-medium;
- Material, size, headspace, fill volume: glass, 150 mL glass beakers containing 100 mL test medium
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): no renewal
- No. of animals per concentration: 20
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Volume: 1 mL/L of Scedenesmus subspicatus
- Density: 10^8 cells/mL


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: due to the light-sensitivity of the test substance, the test was carried out in the dark


TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations of range-finder: 0.01, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
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:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
As the test substance was unsoluble in water, a vehicle was used to homogenously distribute the test substance at all test concentrations. Despite the use of a detergent (Tween 80), a slight turbidity as well as precipitation of the test substance was observed in all test concentrations throughout the duration of the test.
This posed problems when sampling for the water analysis. Samples from all test concentrations were analysed for the test item. The analysis showed that the substance was present in all test concentrations at test initiation. At test end, the test substance was proven to be present only at the highest concentration tested (100 mg/L), although with a very low recovery. This finding could be related to the physicochemical properties of the substance, which precipitated to a high degree when diluted with test medium. However, continuous exposure of daphnids to the highest concentration was considered to have been maintained through the test within the limitations of solubility of the test item.
No product-related effects on daphnids were recorded throughout the exposure up to test concentrations of 100 mL. One single immobilised daphnid occurred at concentrations of 10 and 32 mg test item /L, respectively, but none at higher test concentrations. Consequently, these findings were considered to be non-product related. As the test substance was found to be present at its highest applied concentration throughout the test, the effect concentrations were determined on the base of nominal concentrations.

Any other information on results incl. tables

Table 1: Number of immobilised daphnids after 24 h of exposure

Conc. Nominal (mg/L)

Number of immobilised daphnids after 24 h of exposure

 

1

2

3

4

Total

%

Blank

0

0

0

0

0

0

Vehicle

0

0

0

0

0

0

10

0

0

1

0

1

5

18

0

0

0

0

0

0

32

0

0

1

0

1

5

58

0

0

0

0

0

0

100

0

0

0

0

0

0

 

 Table 2: Number of immobilised daphnids after 48 h of exposure

Conc. Nominal (mg/L)

Number of immobilised daphnids after 48 h of exposure

 

1

2

3

4

Total

%

Blank

0

0

0

0

0

0

Vehicle

0

0

0

0

0

0

10

0

0

1

0

1

5

18

0

0

0

0

0

0

32

0

0

1

0

1

5

58

0

0

0

0

0

0

100

0

0

0

0

0

0

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test results showed that the test item had no effects on daphnids up to nominal concentrations of 100 mg/L. The EC50 (after 48 hr) was determined to be >100 mg/L based on the nominal concentration. Due to the low water solubility of beta-Carotene, precipitation of the test substance was observed throughout the study. The actually dissolved concentrations were considerable below nominal concentrations. The EC50 was > 3.23 mg/L based on the measured concentrations at study initiation and finalisation.
Executive summary:

The purpose of the study was to estimate the acute toxicity (EC50, 48 hr) of the test item to the representative cladoceran species Daphnia magna STRAUS under static conditions. The test was performed in accordance with OECD TG 202.

At test initiation, 20 daphnids were exposed under static conditions to each of the desired test concentrations, the blank vehicle and the blank test medium, respectively. Test concentrations of nominal 10, 18, 32, 58 and 100 mg beta-Carotene were selected based on the results of a pre-test run prior to test initiation at concentrations ranging from 0.01 to 100 mgL. For each concentration and the controls four replicates were performed. Each replicate consisted of 5 specimens in 100 ml test medium (M4-medium). The daphnids were observed for immobilization and behavioural changes after 24 h and 48 hours of exposure to the test substance in the dark.

Samples from all test concentrations were analyzed for beta-Carotene. The analysis showed that the test substance was present in all test concentrations at test initiation. At test end, the test substance was proven to be present only at the highest concentration tested (100 mg/L), although with a very low recovery. This finding could be related to the physico-chemical properties of beta-Carotene which precipitated to a high degree when diluted with test medium. However, continuous exposure of daphnids to the highest test concentration was considered to have been maintained throughout the test within the limitations of solubility of the test substance.

No product-related effects on daphnids were recorded throughout the exposure up to test concentrations of 100 mg/L. One single immobilized daphnid occurred at concentrations of 10 and 32 mg beta-Carotene/L, respectively, but none at higher test concentrations. Consequently, these findings were considered to be non-product related. As the test substance was found to be present at its highest applied concentration throughout the test, the effect concentrations were determined on the base of nominal concentrations.

The test results showed that beta-Carotene had no effects on daphnids up to nominal concentrations of 100 mg/L. The EC50 (after 48h) was determined to be >100 mg/L.