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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:
26 September 2017 to 05 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Samples (approximately 150 mL) of the range-finding and definitive test solutions and controls were collected into Teflon® capped glass bottles (250 mL, no preservative).
- Samples were collected immediately prior to test commencement (0 hour) and at test termination (48 hour, from pooled replicates) for analysis.
- All samples were refrigerated until transported to ISI and kept refrigerated by ISI until analysis was conducted.
- All samples from the range-finding and definitive tests were analysed within 24 hours of sample collection.
Vehicle:
no
Details on test solutions:
- Individual test solutions of the test material for both range-finding and definitive tests were prepared from stock solutions (1000 mg/L nominal concentration) prepared in dilution water without the use of any solubilising agent.
- Stock solutions were prepared in glass volumetric flasks and stirred until the test material was visibly dissolved.
- All stock and test solutions were corrected for purity of the test material (i.e., 80% test material, 20% water).
- All range-finding and definitive tests included a negative control consisting of dilution water with no test material added.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: STRAUS
- Source: The organisms were originally obtained from the Ministry of the Environment and Climate Change (Toronto, Ontario, Canada). The cultures have continuously been actively reproducing in the testing laboratory site since 1988.
- Age of test organisms: First instar, neonate (<24 hours old)
- Feeding during test: no

CULTURE CONDITIONS
- Water used for culturing and testing was the same.
- Feeding frequency: Once/day: 1) yeast, Cerophyll broth, Trout Chow (YCT) and 2) Pseudokirchneriella subcapitata algae
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
200 - 210 mg/L as CaCO3
Test temperature:
20 ± 2°C
pH:
8.3 - 8.5
Nominal and measured concentrations:
Nominal: 110 mg/L
Measured: 104.7 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Beaker
- Material, size, headspace, fill volume: Glass, 75 mL (min)
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 15 mL/ daphnid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used testing was moderately hard groundwater (~350 mg/L as CaCO3) obtained from an aquifer in Aberfoyle, Ontario, Canada. For culture and testing purposes with D. magna, the groundwater was filtered through a 20-micron cellulose acetate filter, diluted with Reverse Osmosis (RO) water to achieve a hardness of ~200 mg/L (as CaCO3). A continuous supply of oil-free compressed air was provided to bring the pH and concentrations of dissolved oxygen and other gases into equilibrium with air. The concentration of dissolved oxygen in the water was maintained at > 80% of the air saturation value.
- Alkalinity: 130 mg/L (as CaCO3)
- Ca/mg ratio: 1.86:1
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water quality parameters (including dissolved oxygen, pH, and conductivity) were measured in the control and all test solutions at the beginning and end of the test. Temperature was monitored and recorded daily. Water hardness was measured at the beginning of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light, 8-hour dark (with 30 minute transition period)
- Light intensity: Ambient laboratory illumination (400 to 800 lux)

EFFECT PARAMETERS MEASURED
- For all test and controls, daphnid immobility was determined after 24 and 48 hours. A test organism was considered to be immobile if it was unable to swim within 15 seconds following gentle agitation of the test solution and/or unable to free itself after gentle tapping of the test vessel.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10, and 100 mg/L.
- A total of 10 daphnids (i.e., 2 replicates with 5 daphnids each) were exposed to each test level under static test conditions. All test organisms were transferred to fresh test and the negative control solutions using a pipette and bulb.
- Results used to determine the conditions for the definitive study: Results of the range-finding test indicated that the 48-hour EC50 for daphnid immobilisation was > 100 mg/L, nominal concentration (i.e., the highest concentration tested). As such, the definitive test was conducted as a Limit Test at a nominal concentration of 110 mg/L, to ensure that 100 mg/L would be maintained during the test period. The 110 mg/L test concentration was prepared by adding 110 mL of a nominal 1000 mg/L stock solution into a 1000 mL volumetric flask and making this up to volume in dilution water.
Reference substance (positive control):
yes
Remarks:
Sodium chloride
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 104.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 104.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
104.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 104.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST
- Confirmation of Exposure Concentrations: Nominal, measured and Time-Weighted Mean (TWM) concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 1. Measured concentrations were similar to nominal concentrations and the beginning and end of the test showing the test material was stable during the 48 hour test period. Nominal test solutions of 1 and 0.1 mg/L were not analysed based on results of a preliminary (non-GLP) test that showed no toxicity to D. magna at concentrations up to and including 100 mg/L.
- Immobility: The range-finding test met all of the test validity criteria. The percentage immobility of daphnids exposed to the test material during the range-finding test along with the nominal and TWM concentrations are given in Table 2. Results of the range-finding test showed that no immobility (0%) of D. magna was observed at concentrations of the test material up to and including 100 mg/L. The TWM concentration corresponding to the nominal 100 mg/L concentration of the test material was 101.4 mg/L.

DEFINITIVE TEST
- Confirmation of Exposure Concentrations: Nominal, measured and TWM concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 3. The TWM concentration corresponding to the nominal 110 mg/L concentration of the test material was 104.7 mg/L.
- Immobility: The definitive test met all of the test validity criteria. The definitive test was conducted as a Limit Test at a single concentration of the test material (i.e., 110 mg/L). A summary of the test results expressed in terms of the percent daphnid immobility observed in the test after 24 and 48 hours is summarized in Table 4. Results of the definitive test confirmed the results of the range-finding test showing 0% immobility of daphnids at a TWM concentration of 104.7 mg/L. The 24 and 48-hour EC50s for immobility of daphnids were reported to be greater than the TWM concentration of 104.7 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were reported to be 104.7 and >104.7 mg/L, respectively.

TEST VALIDITY CRITERIA
All of the validity criteria for the definitive tests were met as listed below:
- Immobility of the controls did not exceed 10%.
- Dissolved oxygen concentrations at the end of the test were ≥ 3 mg/L in the control and test solutions.
Results with reference substance (positive control):
- Sodium chloride was the reference toxicant used in this study. The LC50 was compared to historical data and was deemed acceptable based on results falling within the 95% confidence interval (warning limit) calculated for the last twenty (20) reference tests.
- In conjunction with the definitive test, the 48-hour LC50 for the Daphnia magna reference toxicant test was 6.2 g/L.
Reported statistics and error estimates:
- All test results were based on Time-Weighted Mean (TWM) concentrations of the test material.
- Estimates of the 24 and 48-hour EC50 from the range-finding and definitive test results were based on visual inspection of the data, since during the study, no immobility of daphnids was observed at any concentration up to and including 100 mg/L (nominal).

Table 1: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material for the Range-Finding Test

Nominal Concentration (mg/L)

Measured Concentration (mg/L)

Time-Weighted Mean Concentration (mg/L)

0 Hours

48 Hours

100

101.13

99.59

101.4

10

10.18

10.33

10.3

1

N/A

N/A

N/A

0.1

N/A

N/A

N/A

0 (Negative Control)

<MDL

<MDL

<MDL

N/A = not analysed

MDL = 0.5 mg/L

Table 2: Immobility of D. magna After 24 and 48 Hours Exposure to the Test Material During the Range-Finding Test

Nominal Concentration (mg/L)

Time-Weighted Mean (mg/L)

Immobility (%)

T= 24 hours

T= 48 hours

100

101.4

0

0

10

10.3

0

0

1

N/A

0

0

0.1

N/A

0

0

0 (Negative Control)

<MDL

0

0

N/A = not analysed

MDL = 0.5 mg/L

Table 3: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material for the Definitive Test

Nominal Concentration (mg/L)

Measured Concentration (mg/L)

Time-Weighted Mean Concentration (mg/L)

0 Hours

48 Hours

110

104.2 and 105.4

104.8 and 104.2

104.7

0 (Negative Control)

<MDL

<MDL

<MDL

 MDL = 0.5 mg/L

Table 4: Immobility of D. magna After 24 and 48 Hours Exposure to the Test Material During the Definitive Test

Nominal Concentration

(mg/L)

Time-Weighted Mean

Concentration (mg/L)

Immobility (%)

T = 24 hours

T = 48 hours

110

104.7

0

0

0 (Control)

<MDL

0

0

MDL = 0.5 mg/L

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 24 and 48-hour EC50s were reported to be >104.7 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were reported to be 104.7 and >104.7 mg/L, respectively.
Executive summary:

The aquatic toxicity of the test material was investigated in accordance with the standardised guideline OECD 202, under GLP conditions.

An acute immobilisation test was performed on Daphnia magna. The test was conducted over a 48-hour test period under static test conditions.

Range-finding and definitive tests both met all the test validity criteria. Test results were based on measured concentrations, or more specifically, Time-Weighted Mean (TWM) concentrations. All test solutions and results were corrected for purity of the test material (i.e., 80% test material; 20% water).

Results of the range-finding test indicated that the 48-h EC50 for immobility was >101.4 mg/L. As such, the definitive test was conducted as a Limit Test at a single nominal concentration of the test material of 110 mg/L to ensure that a concentration of ≥ 100 mg/L was maintained throughout the test period. The TWM concentration of test solution was 104.7 mg/L. Results of the definitive test showed that there was no immobility of daphnids in the study.

Under the conditions of this study, the 24 and 48-hour EC50s were reported to be >104.7 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were reported to be 104.7 and >104.7 mg/L, respectively.

Description of key information

Under the conditions of the study, the 24 and 48-hour EC50s were reported to be > 104.7 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were reported to be 104.7 and >104.7 mg/L, respectively.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
104.7 mg/L

Additional information

The aquatic toxicity of the test material was investigated in accordance with the standardised guideline OECD 202, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

An acute immobilisation test was performed on Daphnia magna. The test was conducted over a 48-hour test period under static test conditions.

Range-finding and definitive tests both met all the test validity criteria. Test results were based on measured concentrations, or more specifically, Time-Weighted Mean (TWM) concentrations. All test solutions and results were corrected for purity of the test material (i.e., 80% test material; 20% water).

Results of the range-finding test indicated that the 48-h EC50 for immobility was >101.4 mg/L. As such, the definitive test was conducted as a Limit Test at a single nominal concentration of the test material of 110 mg/L to ensure that a concentration of ≥ 100 mg/L was maintained throughout the test period. The TWM concentration of test solution was 104.7 mg/L. Results of the definitive test showed that there was no immobility of daphnids in the study.

Under the conditions of this study, the 24 and 48-hour EC50s were reported to be > 104.7 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were reported to be 104.7 and > 104.7 mg/L, respectively.