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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:
11 - 29 Jun 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Remarks:
GC
Details on sampling:
- Concentrations: 0 (control), 2.6, 6.4, 16, 40 and 100% v/v saturated solution
- Sampling method: Samples were taken from the freshly prepared control and each test group from the bulk test preparation at 0 and 24 hours and from the old test media (pooled replicates) at 24 and 48 hours for immediate quantitative analysis. Duplicate samples were taken at 24 and 48 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: The samples were analyzed on the day of sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals. The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 4.3 mg/L could be obtained using a saturated solution method of preparation. For more details see field "any other information on material and methods incl. tables".
- Method: Four nominal amounts of test item (27.8 mg) were dispersed under the surface of 555 mL of test water in completely filled glass conical flasks sealed with ground glass stoppers. The flasks prepared for the test media renewal at 24 hours were additionally sealed with Parafilm tape. All flasks were shaken in an incubator at approximately 150 rpm at a temperature of 21°C for 24 hours. After the shaking period, the replicates were pooled and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (the first approximate 100 mL used to pre-condition the filter was discarded), to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations.
- Differential loading: no
- Controls: Culture medium only
- Calculation of mean measured concentrations: The geometric mean measured concentrations for each period of media renewal (0-24 and 24-48 hours) were calculated and the arithmetic mean of the two values determined. Where a measured concentration of less than the LOQ of the analytical method was determined, a value of half the LOQ was substituted into the equation.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: in-house laboratory cultures
- Feeding during test: no
- Culturing: Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Pseudokirchneriella subcapitata) and GEMMA Micro 300 fishfood suspension. Culture conditions ensured that reproduction was by parthenogenesis.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Study design

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

Test conditions

Test temperature:
20°C - 21°C
pH:
7.9 - 8.1
Dissolved oxygen:
8.4 - 9.0 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 2.6, 6.4, 16, 40 and 100% v/v saturated solution
Geometric mean measured concentrations: < LOQ (control), 0.06, 0.12, 0.34, 0.91, 3.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass vessels
- Type: closed, sealed with ground glass stoppers
- Fill volume: 150 mL
- Aeration: no
- Renewal rate of test solution: test media renewal after 24 hours
- 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: Elendt M4 medium according to the OECD guideline 202
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water temperature, dissolved oxygen concentrations and pH were recorded at the start of the test, on both old and new media at 24 hours and at termination of the test. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 hours light/dark
- Light intensity: 1145 - 1179 lux

EFFECT PARAMETERS MEASURED: Immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.#

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: No immobilization was observed at the test concentration of 10% v/v saturated solution, however; 10% immobilization was observed at test concentrations of 0.10 and 1.0% v/v saturated solution. This was considered to be non concentration dependent mortality. At the test concentration of 100% v/v saturated solution, 100% immobilization was observed.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Due to the nature of the data it was not possible to calculate the slope and error of response curve.
Details on results:
Other biological observations: Sub-lethal effects of exposure were observed in test concentrations of 0.34 and 0.91 mg/L saturated solution. These responses were reduced mobility and pale appearance (for more details see "any other information on results incl. tables".
- Mortality of control: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, at the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
Results with reference substance (positive control):
A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test.
- Test concentrations: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- Dose-response test: yes
- EC50 (24 h): 1.1 mg/L (95% confidence limits: 0.95 - 1.3 mg/L), EC50 (48 h): 0.80 mg/L (95% confidence limits: 0.12 - 1.5 mg/L)
- Other: NOEC: 0.56 mg/L, LOEC: 1.0 mg/L
Reported statistics and error estimates:
The EC50 value at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Any other information on results incl. tables

Table 1: Effect concentrations after 24 hours

 

Time Point (Hours)

 

EC50

(mg/L)

 

95% Confidence Limits (mg/L)

 

NOEC

(mg/L)

 

LOEC

(mg/L)

24

1.6

*

0.91

3.0

Table 2: Analytical results.

Nominal concentration of the test item in samples

(% v/v saturated solution)

Determined concentration of the test item

(mg/L)

Geometric mean (mg/L)

0 hours

24 hours

(old)

24 hours 

(fresh)

48 hours

(old)

Control

 Not detected

Not detected

 Not detected

Not detected

 -

2.6

 < LOQ

 < LOQ

< LOQ

 < LOQ

0.06

6.4

0.155

0.138

0.122

 < LOQ

0.12

16

0.449

0.397

0.338

0.185

0.34

40

1.19

1.11

0.962

0.533

0.91

100

3.52

3.41

3.28

1.93

3.0

Table 3: Cumulative immobilization data and observations in the acute test with D. magna after 24 hours. 

Geometric Mean Measured Concentration

(mg/L)

24 hours

Cumulative Immobilized Daphnia

(Initial Population: 5 per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.060

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.12

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.34

0

0

0

0

0

0

1 R 4 N

5 N

5 N

5 N

0.91

0

0

0

0

0

0

5 N

5 N

2 R 3 N

1 R 1 P

3 N

3.0

5

5

5

5

20

100

AI

AI

AI

AI

Rep = Replicate

N= Normal

R =Reduced mobility

P = Pale

AI = All daphnids immobilized

Table 4: Cumulative immobilization data and observations in the acute test with D. magna after 48 hours.

Geometric Mean Measured Concentration

(mg/L)

48 hours

Cumulative Immobilized Daphnia

(Initial Population: 5 per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.060

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.12

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.34

0

0

0

0

0

0

1 R 4 N

5 N

5 N

5 N

0.91

0

0

0

0

0

0

2 R 3 N

1 R 4 N

2 R 3 N

3 R 2 N

3.0

5

5

5

5

20

100

AI

AI

AI

AI

Rep = Replicate

N= Normal

R =Reduced mobility

P = Pale

AI = All daphnids immobilized

Table 5: Validity criteria for OECD 202.

Criterion from the guideline

Outcome

Validity criterion fulfilled

In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.

 0%

 yes

The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.

 8.4 - 9.0 mg/L

 yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.