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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:
24 January 2001 to 26 January 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
Version / remarks:
Adaptation of tests solutions for poorly soluble substances
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Using syringes, samples of the test material were each separately added onto the surface of reconstituted water (2 litres) to give the required loading rates. Initially each vessel was stirred by magnetic stirred using a stirring rate giving a vortex depth extending from the surface to approximately 5 % of the distance to the bottom of the vessel. However, at the start of the first range finding test, the stirring of the test material was observed to form stringy particles which began to break up. The the stirring rate was adjusted to approximately 30 rpm to minimise gross mixing of the test material. The test solutions were stirred for 24 hours. After stirring, the solutions were allowed to settle for 2 hours.
A glass tube, covered at one end with parafilm was then inserted into each vessel and sealed down to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was then inserted into the glass tube and pushed through the parafilm seal. At the opposite end of the tube, a glass wool plug was inserted (approximately 2-4 cm in length) to rap any micro-emulsions. Prior to use, the glass wool was preconditioned by thoroughly soaking overnight in deionised water, rinsing three times with deionised water and reconstituted water followed by autoclaving. The aqueous phase or WAF was then removed by siphon (the first 75-100 mL discarded) and used for testing. The test solutions were prepared in the same way for the range finding studies and the definitive test.
- Controls: Untreated control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to siphoning, the test solutions were observed to have stringy globules at the surface of the water. After 24 hours stirring and 2 hours settling the loading rates up to 5.6 mg/L had stingy globules at the surface of the water. At 10-100 mg/L, the test solutions were observed to be cloudy turbid dispersions with stringy globules at the surface of the water. The test solutions were siphoned after settling. After siphoning the test solutions up to and including 5.6 mg/L were clear colourless solutions. At concentrations greater than or equal to 10 mg/L, the test solutions were observed to be very pale white turbid dispersion with increasing turbidity with increasing concentration. No changes were observed in the test solutions during testing. Further siphoning was not found to reduce the amount of undissolved test material.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In house culture (1st instar)
- Feeding during test No feeding performed during test
- Food type: Chlorella sp. algal suspension
- Frequency: Daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature-controlled room at 21 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours dark cycle with a 20-minute dusk/dawn transition. Culture conditions ensured reproduction by parthenogenesis. Gravid adults were isolated the day before initiation of the test. Young daphnids produced overnight were less than 24 hours old were removed for testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Theortical - 250 mg/L as CaCO3
Test temperature:
Approximately 21 °C
pH:
7.8 ± 0.2
Dissolved oxygen:
7.9-8.4 mg O2/L
Conductivity:
< 5 µS/cm
Nominal and measured concentrations:
Nominal loading rates:
0, 1, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type (delete if not applicable): Closed (covered to prevent evaporation)
- Material, size, headspace, fill volume: 250 mL glass jars filled to approximately 200 mL
- Aeration: Test solutions were not aerated during the test. Reconstituted water was aerated to saturation prior to use.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
25 mL of each of the following added to deionised water (to make a total volume of 1 L)
11.76 g/L CaCL2.2H2O
4.93 g/L MgSO4
2.59 g/L NaHCO3
0.23 g/L KCl
- Conductivity: < 5 µS/cm
- Culture medium different from test medium: Adult Daphnia maintained in reconstituted water.

OTHER TEST CONDITIONS
- Adjustment of pH: the pH was adjusted with NaOh or HCl where necessary to give a pH of 7.8 ± 0.2.
- Photoperiod: 16 hours light, 8 hours darkness with a 20 minute dusk/dawn period

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daphnia were considered to be immobilised if they were unable to swim for 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: 1st Range finder: 0 (control), 100, 100 (double filtered), 1000 and 1000 (double filtered) mg/L
2nd Range finder: 0 (control), 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: During the initial range finding study, 100 % immobilisation was observed at each of the 100 and 100 mg/L loading rates after 24 hours of exposure. As each of these WAFs was observed to be a turbid white dispersion microscopic examination was performed on the immobilised daphnia. They were observed to be free of the test material. The effects were therefore considered to be indicative of toxicity and not a physical effect.
The results of the second range finding study indicated no immobilisation at 1 mg/L and 100 % at 10 and 100 mg/L.
The definitive test concentrations were selected based on the result of the two range finding tests. The test solutions for the definitive test were prepared by employing a single phase separation method. A single siphon through one glass wool plug was utilised as double filtration did not significantly reduce the amount of undissolved test material.
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
5.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
immobilisation
Remarks on result:
other: 95 % 5.1-6.6
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
4.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
immobilisation
Remarks on result:
other: 95 % CI 3.2-5.6
Key result
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
immobilisation
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
immobilisation
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Concentrations exceeding 5.6 mg/L had evidence of undissolved test material. To ensure that the effects were due to toxicity, and not physical effects of undissolved test material, all immobilised daphnids were examined microscopically. The daphnids were free of test material at examination.
Reported statistics and error estimates:
The EL50 values and confidence limits at 24 hours were calculated by Spearman-Karber method of Hamilton et al (1977) Trimmed Spearman-Karber Method for Estimating Median Lethal Concentration in Toxicity Bioassays Environ Sci Technol 11 (7): 714-719, using the ToxCalc software package and at 48 hours the EL50 was calculated using the geometric mean method using the following equation:
EL50 value = √L2 x L2
Where L1 = loading rate showing 0 % immobilisation
L2 = loading rate showing 100 % immobilisation
When only one partial response was observed, the trimmed Spearman-Karber method is appropriate.
If there is no immobilisation between 0 and 100 % immobilisation, then the geometric mean of the highest loading rate showing no lethality and the lowest loading rate showing 100 % lethality was calculated. The loading rates resulting in 0 % and 100 % immobilisation are the 95 % confidence limites.

Table 1: Initial Range Finding Test Results

Nominal Loading Rate
(mg/L)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours*

Control

0

100

10

100 (double filtered)

10

1000

10

1000 (double filtered)

10

* Terminated at 24 hours due to 100 % mortality.

 

Table 2: Final Range Finding Test Results

Nominal Loading Rate
(mg/L)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

1.0

0

0

10

10

10

100

10

10

 

Table 3: Definitive Test Results

Nominal Loading Rate
(mg/L)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

1.0

0

0

0

0

0

0

0

0

1.8

0

0

0

0

0

0

0

0

3.2

0

0

0

0

0

0

0

0

5.6

3

6

9

45

10

10

20

100

10

10

10

20

100

10

10

20

100

18

10

10

20

100

10

10

20

100

32

10

10

20

100

10

10

20

100

56

10

10

20

100

10

10

20

100

100

10

10

20

100

10

10

20

100

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the acute toxicity of the test material to the freshwater invertebrate Daphnia magna was determined to be EL50 4.2 mg/L (loading rate WAF) with 95 % confidence limits of 3.2-5.6 mg/L loading rate WAF. The No Observed Effect Loading Rate at 48 hours was 3.2 mg/L loading rate WAF.
Executive summary:

The acute toxicity of the test material to the aquatic freshwater invertebrate Daphnia magna, was investigated in accordance with the methods OECD 202, EU Method C.2 and EPA OPPTS 850.1010.

Following the preliminary range finding tests, 20 daphnids were exposed to WAFs of the test material up to 100 mg/L under static conditions.

Under the conditions of the test, the acute toxicity of the test material to the freshwater invertebrate Daphnia magna was determined to be EL50 4.2 mg/L (loading rate WAF) with 95 % confidence limits of 3.2-5.6 mg/L loading rate WAF. The No Observed Effect Loading Rate at 48 hours was 3.2 mg/L loading rate WAF.

Description of key information

The acute toxicity of the test material to the aquatic freshwater invertebrate Daphnia magna, was investigated in accordance with the methods OECD 202, EU Method C.2 and EPA OPPTS 850.1010.

Following the preliminary range finding tests, 20 daphnids were exposed to WAFs of the test material up to 100 mg/L under static conditions.

Under the conditions of the test, the acute toxicity of the test material to the freshwater invertebrate Daphnia magna was determined to be EL50 4.2 mg/L (loading rate WAF) with 95 % confidence limits of 3.2-5.6 mg/L loading rate WAF. The No Observed Effect Loading Rate at 48 hours was 3.2 mg/L loading rate WAF.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
4.2 mg/L

Additional information