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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:
07 September 2017 to 14 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes
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 the same day that the samples were collected.
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.
- Due to the low solubility of the test material, stock solutions were prepared in 1 or 2-L glass aspirator bottles, stirred for approximately 23 hours at a rate sufficient to maintain a vortex between approximately 10 - 35% of the solution depth using a stir bar and stir plate. The solutions were then settled for approximately 1 hour. For the definitive test, the first ~ 100 mL of stock solution removed from the glass aspirator was discarded. The individual test solutions were prepared by adding an appropriate amount of the stock solution into a 500 mL volumetric flask and making this up to volume with dilution water.
- All tests (range-finding and definitive) 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: Daphnia magna STRAUS
- Source: The organisms were originally obtained from the Ministry of the Environment and Climate Change (Toronto, Ontario, Canada), the organisms were cultured continuously from a population that has been actively reproducing in the testing laboratory since 1988.
- Age: First instar, neonate (< 24 hours old)
- Feeding during test: no

CULTURE CONDITIONS
- Test organism acclimation was not required, since test organisms were cultured under conditions identical to those of the test.
- Type and amount of food: Once/day: yeast, Cerophyll broth, Trout Chow and Pseudokirchneriella subcapitata algae
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
200 mg/L as CaCO3
Test temperature:
20 ± 2 °C
pH:
8.0 – 8.5
Nominal and measured concentrations:
Nominal: 62.5, 125, 250, 500 and 1,000 mg/L
Measured: 0.5, 1.0, 2.0, 4.3 and 8.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beaker, 100 mL
- Material, size, headspace, fill volume: 75 mL (min)
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Loading rate: 15 mL/daphnid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The source of water used for D. magna culturing and 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.
- 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
- Adjustment of pH: no
- Photoperiod: 16-hour light, 8-hour dark (with 30 minute transition period)
- Light intensity: 400 to 800 lux (Ambient laboratory illumination)

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: 1.0, 10, 100 and 1000 mg/L
- Concentrations were prepared by adding 0.05, 0.5, 5, and 50 mL of a nominal 1000 mg/L stock solution, respectively into a 500 mL volumetric flask and making this up to volume with dilution water.
- A total of 10 daphnids (i.e., 2 replicates with 5 daphnids each) were exposed to each test level under static test conditions (i.e., no renewal of the test solution). 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 between 100 and 1000 mg/L nominal concentration of the test material. As such, the definitive test was conducted as a multiple concentration test. Nominal concentrations of 62.5, 125, 250, 500 and 1,000 mg/L, were prepared by adding 31.25, 62.5, 125, 250, and 500 mL of a nominal 1000 mg/L stock solution, respectively into a 500 mL volumetric flask and making this up to volume in dilution water.
Reference substance (positive control):
yes
Remarks:
Sodium chloride
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
443 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % C.L: 330-601 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
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 below nominal concentrations due to the low solubility of the test material. Nominal concentrations of 1000 and 100 mg/L achieved TWM concentrations of 11.28 and 0.87 mg/L, respectively (i.e., approximately 1% of the nominal concentration).
- Immobility: The range-finding test met all 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 100% immobility of D. magna after 48 hours of exposure to 1000 mg/L, while all lower nominal concentrations (i.e., ≤ 100 mg/L) showed no effect. As such, the definitive test was conducted as a multi-concentration test, with the EC50 expected to be in the range of 100 to 1000 mg/L.

DEFINITIVE TEST RESULTS
- 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 of 8.2 mg/L was achieved from preparation of the nominal 1000 mg/L stock solution of the test material used for the highest concentration of the definitive test. The spike recovery was 80.1 to 83.8% which was acceptable (average > 80%). The measured concentrations were lower at test end as compared to test start.
- Immobility: The definitive tests met all of the test validity criteria. A summary of the test results expressed in terms of the percent daphnid immobility observed in the test after 24 and 48 hours is summarised in Table 4. Results of the definitive test showed that mobility of daphnids was significantly reduced at nominal concentrations ≥ 500 mg/L. Although daphnid immobility at lower concentrations ranged from 5 to 20%, the results were not significantly different from the controls. The 24-EC50 for immobility of daphnids was reported >1000 mg/L as there was no (0%) immobility of daphnids observed at any concentration during this period. The 48-hour EC50 was estimated to be 443 mg/L (95% confidence limits 330 to 601 mg/L). NOEC and LOEC for immobility of daphnids were reported to be 250 and 500 mg/L, respectively.
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 reference tests.
- In conjunction with the definitive test, the 48-hour LC50 for the Daphnia magna reference toxicant test was 6.1 g/L.
Reported statistics and error estimates:
- All endpoints were generated using nominal concentrations of the test material at test initiation (t=0h). Although analytical concentrations were verified, due to the nature of the test material (UVCB), measured concentrations were not necessarily representative of the whole substance. Therefore, statistical analysis and end points are expressed here in terms of nominal concentrations.
- The 24-EC50 for the definitive test was reported as a greater than value as no immobility (0 %) of daphids was observed at any concentration. The 48- hour EC50 (and 95 % confidence limits) for the definitive test were estimated using Linear Regression (MLE) (CETIS, 2013). The 48- hour NOEC and LOECs were determined using Fisher Exact Test (with adjustment using the
Bonferroni-Holm) test (CETIS, 2013).

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)

T = 0 Hours

T = 72 Hours

1000

18.00d

6.48e

11.28

100

1.19

0.61

0.87

10

0.17

N/A

0.17g

1

< MDL

N/A

< MDL

0 (Negative Control)

< MDL

< MDL

< MDL

d Average of duplicate analyses. The Relative Percent Difference (RPD) = 2.3%

e Average of duplicate analyses. RPD = 14.3%

MDL (Method Detection Limit) = 0.1 mg/L

g Based on a single analysis (not a TWM)

N/A = not analysed

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)

Immobility (%)

T= 24 hours

T= 48 hours

1000

0

100

100

0

0

10

0

0

1

0

0

0 (Negative Control)

0

0

 

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)

T=0 Hours

T=48 Hours

1000

9.86d

6.74e

8.2

500

5.23

3.41

4.3

250

2.56

1.52

2.0

125

1.15

0.79

1.0

62.5

0.41

0.57

0.5

0 (Negative Control)

< MDL

< MDL

< MDL

d Result of duplicate analysis. RPD 8.6%

e Result of duplicate analysis. RPD 4.8%

Method Detection Limit (MDL) = 0.1 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)

Measured Concentration (mg/L)

T=24 Hours

T=48 Hours

1000

0

85

500

0

60

250

0

20

125

0

5

62.5

0

5

0 (Negative Control)

0

0

TEST VALIDITY CRITERIA

All of the validity criteria for the definitive tests were met:

- 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.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 24-hour EC50 based on results of the definitive test were reported to be > 1000 mg/L. The 48-hour EC50 (95% Confidence Limits) was estimated to be 443 (330 – 601) mg/L. All EC50s exceeded the OECD limit of 100 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 250 mg/L and 500 mg/L, respectively.
Executive summary:

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

Both the range-finding and definitive tests met all the test validity criteria.

Test solution preparation included continuous stirring for approximately 23 hours, followed by a settling period of approximately 1 hour. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only.

Results of the range-finding test indicated that the 48-h EC50 for immobility of D. magna based on nominal concentrations of the test material was in the range of 100 mg/L to 1000 mg/L. As such, the definitive test was conducted as a multiple-concentration test.

Under the conditions of this study, the 24-hour EC50 based on results of the definitive test were reported to be > 1000 mg/L. The 48-hour EC50 (95% Confidence Limits) was estimated to be 443 (330 – 601) mg/L. All EC50s exceeded the OECD limit of 100 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 250 mg/L and 500 mg/L, respectively.

Description of key information

Under the conditions of this study, the 24-hour EC50 based on results of the definitive test were reported to be >1000 mg/L. The 48-hour EC50 (95 % Confidence Limits) was estimated to be 443 (330 – 601) mg/L. All EC50s exceeded the OECD limit of 100 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 250 mg/L and 500 mg/L, respectively.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
443 mg/L

Additional information

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

Both the range-finding and definitive tests met all the test validity criteria (i.e., immobility ≤ 10 % in the control; dissolved oxygen concentration at test termination ≥ 3 mg/L in all control and test vessels).

Test solution preparation included continuous stirring for approximately 23 hours, followed by a settling period of approximately 1 hour. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only.

Results of the range-finding test indicated that the 48-h EC50 for immobility of D. magna based on nominal concentrations of the test material was in the range of 100 mg/L to 1000 mg/L. As such, the definitive test was conducted as a multiple-concentration test.

Under the conditions of this study, the 24-hour EC50 based on results of the definitive test were reported to be >1000 mg/L. The 48-hour EC50 (95 % Confidence Limits) was estimated to be 443 (330 – 601) mg/L. All EC50s exceeded the OECD limit of 100 mg/L. The 48-hour NOEC and LOEC for immobility of daphnids were estimated to be 250 mg/L and 500 mg/L, respectively.