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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:
2017-09-19 to 2017-12-07
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
Principles of method if other than guideline:
There were no deviations and no amendments to this study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch/Lot no: 160306
Purity: >99%

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: approximately 40 mL of each test solution and the negative control were collected in glass containers of appropriate size
- Sample storage conditions before analysis: stored refrigerated (2°C to 8°C) until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a stock solution was prepared at 100 mg test item/L and test solutions were prepared as dilutions of this stock
- Controls: negative control (in parallel), positive control (in a separate non-GLP study)
- Evidence of undissolved material: no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: Aquatic BioSystems (Fort Collins, CO, USA), in-house culture
- Age of parental stock: less than 24 hours old
- Feeding during test: no; only during culturing
- Food type: Pseudokirchneriella subcapitata and 1.0 mL of yeast-cereal-grass fermented trout chow
- Amount: 4.0 mL of Pseudokirchneriella subcapitata and 1.0 mL of yeast-cereal-grass fermented trout chow
- Frequency: three times per week



METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnids were randomly selected and transferred to each of the four replicates of the negative control, using a wide-bore glass pipette
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
according to guideline
Post exposure observation period:
no
Hardness:
92 mg/L CaCO3
Test temperature:
19.9 – 20.5 °C
pH:
7.3 – 8.1
Dissolved oxygen:
8.9 – 9.0 mg/L
Salinity:
not applicable
Conductivity:
325 (negative control), 321 (6.25 mg/L), 320 (12.5 mg/L), 319 (25 mg/L), 318 (50 mg/L) and 314 (100 mg/L) µS/cm
Nominal and measured concentrations:
Nominal: 6.25, 12.5, 25, 50, and 100 mg/L
Measured mean concentrations of Tungsten in test solution: 4.1, 8.4, 16.6, 29.7 and 51.6 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type: closed with Plexiglas
- Material, size, headspace, fill volume: glass, 250 mL, 200 mL test volume
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: moderately hard, reconstituted water for culturing
- Particulate matter: no
- Water quality measurements: prior to use
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-h light : 8-h dark with a 30 minute transition period
- Light intensity: 609 – 679 lux

EFFECT PARAMETERS MEASURED
- Immobility

RANGE-FINDING STUDY
- Test concentrations: 0, 0.1, 1.0, 10, and 100 mg test item/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
zinc sulphate heptahydrate (CAS no. 7446-20-0)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
70.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 56.6 - 84.6 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
The 48-hours LC50 and 95% confidence limits for the reference item test are 0.77 mg/L (0.48, 1.60 mg/L). This value indicates that the test organisms responded normally to the reference item and confirms the suitability of the test system.
Reported statistics and error estimates:
The number of immobilised daphnids at the 24 ± 1hours and 48 ±1 hours time points was calculated using Microsoft Excel and graphically plotted against the negative control and nominal test item concentrations to create a concentration response curve. The mean and standard deviation for the negative control and each nominal test item concentration was calculated, where appropriate, as per the OECD 202 Test Guideline.

The 48-hour EC50 with 95% confidence limits (based on nominal concentrations) and the NOEC and LOEC values for immobilisation were calculated using a computerised statistical package, CETIS™ (Version 1.9.2.4), a Comprehensive Environmental Toxicity Information System, recommended by Environment Canada. All reported values were rounded according to the Work Instruction for Significant Figures in Data Reporting (COR WI-00114).

Table 1: Number and percentage of Daphnia immobilised after 24-h exposure

Nominal Concentration of Test Item
(mg/L)

Mean
± SD
(%)

Replicate
A
(# / %)

Replicate
B
(# / %)

Replicate
C
(# / %)

Replicate
D
(# / %)

Negative Control (0)

0 ± 0

0 / 0

0 / 0

0 / 0

0 / 0

6.25

0 ± 0

0 / 0

0 / 0

0 / 0

0 / 0

12.5

0 ± 0

0 / 0

0 / 0

0 / 0

0 / 0

25

0 ± 0

0 / 0

0 / 0

0 / 0

0 / 0

50

0 ± 0

0 / 0

0 / 0

0 / 0

0 / 0

100

0 ± 0

0 / 0

0 / 0

0 / 0

0 / 0

Table 2: Number and percentage ofDaphnia immobilised after 48-h exposure

Nominal Concentration of Test Item
(mg/L)

Mean
± SD
(%)

Replicate
A
(# / %)

Replicate
B
(# / %)

Replicate
C
(# / %)

Replicate
D
(# / %)

Negative Control (0)

0 ± 0

0 / 0

0 / 0

0 / 0

0 / 0

6.25

0 ± 0

0 / 0

0 / 0

0 / 0

0 / 0

12.5

5 ± 10

0 / 0

0 / 0

0 / 0

1 / 20

25

5 ± 10

0 / 0

0 / 0

1 / 20

0 / 0

50

20 ± 23

2 / 40

0 / 0

2 / 40

0 / 0

100

80 ± 16

5 / 100

4 / 80

3 / 60

4 / 80

Table 3: Measured concentrations of Tungsten in test solutions

Nominal concentration of test item [mg/L] Tungsten [mg/L]
Initial Final Mean SD
Negative control (0) BQL BQL N/A N/A
6.25 4.1 4.1 4.1 0.0
12.5 8.2 8.6 8.4 0.3
25 15.4 17.8 16.6 1.7
50 25.5 33.9 29.7 5.9
100 43.2 60.1 51.6 12.0

BQL: Below Quantitation (<0.002 mg/L)

N/A: Not Applicable

Validity criteria fulfilled:
yes

Description of key information

The 48-hour EC50 of the test item was determined to be 70.8 mg/L (based on nominal concentrations).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
70.8 mg/L

Additional information

The toxicity to aquatic invertebrates was assessed in Acute Immobilisation Test with Daphnia magna according to OECD Guideline 202 and in compliance with GLP principles. This static aquatic test was conducted under controlled conditions using less than 24 hours old Daphnia magna neonates over a 48-hour exposure period and was carried out using the following nominal concentrations: 6.25, 12.5, 25, 50, and 100 mg test Item/L, along with a negative control (0 mg/L). At test completion, there were no immobilised or dead daphnids in the negative control or in the 6.25 mg/L test item solutions. The remaining test item solutions had 5% - 80% immobilised or dead daphnids. For confirmation of test item concentrations, Tungsten was analysed as a proxy for the test item. Concentration of Tungsten in test solutions was consistent between test initiation and test completion, with values within ± 20% of the mean Tungsten concentration for each nominal test item concentration. Therefore, the biological test endpoint was calculated using the nominal concentrations of the test item. Based on the nominal concentrations of the test item, the 48-hour EC50 was 70.8 mg/L (95% confidence limits: 56.6 - 84.6 mg/L). The 48-hour NOEC and LOEC was determined to be 50 mg/L and 100 mg/L, respectively. All validity criteria of the guideline were fulfilled.