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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:
25 February 2017 tot 10 March 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:
April 2004
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
No. 440/2008
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Verification of Test Concentrations:
Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Storage:
The samples were analyzed on the day of receipt.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.15 mg/L could be obtained using a saturated solution method of preparation.

The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.

Range-finding test:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100% v/v saturated solution.

A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Feeding during test: Daphnids received no food during exposure and the test vessels were not aerated.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 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 (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
none
Test temperature:
20 - 22 °C
pH:
7.6 - 8.1
Dissolved oxygen:
8.5 - 9.4 mg O2/L
Nominal and measured concentrations:
Range-finding test:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the Limit of Quantification (LOQ) of the analytical method employed, determined to be 0.0030 mg/L, to 0.27 mg/L. Analysis of the test preparations at 24 hours showed that measured concentrations ranged from less than the LOQ to 0.30 mg/L.

No immobilization was observed at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution, however, immobilization was observed at 100% v/v saturated solution. A single daphnia was observed to be immobilized in the control group, however, this was considered to be due to natural causes and as such was considered not to have had an impact on the outcome of the test.

Definitive test:
Based on the results of the range-finding test test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test.

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 0.051 to 0.66 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.039 and 0.60 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

The geometric mean measured test concentrations are presented under "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL completely filled and sealed ground glass stoppered vessels
- Type: test vessels were sealed to reduce evaporation
- Fill volume: approximately 150 mL, test vessels were completely filled and sealed
- Aeration: No
- Type of flow-through: No
- Renewal of test solution: Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (Elendt M7 medium) used for both the range-finding and definitive tests:
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075
The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC.

- Culture medium different from test medium: No, adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: between 200 and 1200 Lux

EFFECT PARAMETERS MEASURED:
Test organism observations: Any 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.

Water quality: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

RANGE-FINDING TEST:
Cummulative immobilization data are presented under "Any other information on materials and methods".
Reference substance (positive control):
yes
Remarks:
potassium dichromate (Conducted between 20 October 2016 and 22 October 2016)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.L.: 0.35 - 0.53 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 0.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given under "Any other information on results incl. tables". Also the results of the positive control are presented there. Exposure conditions for the positive control were similar to those in the definitive test.

- Sub-Lethal effects:
Sub-lethal effects of exposure were observed in the 18, 32, 56 and 100% v/v saturated solution test concentrations. These responses were reduced movement and a pale color (see Any other information on results incl. tables).

- Validation Criteria:
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

- Water Quality Criteria:
Temperature was maintained at 20 oC to 22 oC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The pH was within the range of 7.6 - 8.1 and the oxygen concentrations was within the range of 8.5 - 9.4 mg O2/L.
Throughout the test the light intensity was observed to be in the range 512 to 594 Lux.

- Observations on Test Item Solubility:
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 used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave results presented under "Any other information on results incl. tables"


Reported statistics and error estimates:
The slopes and their standard errors of the response curves from the definite test at 24 and 48 hours were 0.75 (SE = 1.3) and 5.4 (SE = 0.04) respectively.

The geometric mean measured test concentrations in the definite test were determined to be:

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

10

0.052

18

0.084

32

0.16

56

0.27

100

0.62

Cumulative Immobilization Data and Observations in the Definitive Test:

Nominal
Concentration
(% v/v Saturated Solution)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

0

0

0

0

0

5 N

5 N

5 N

5 N

56

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

1

2

3

15

5 P + R

5 P + R

4 P + R

3 P + R

 

Nominal
Concentration
(% v/v Saturated Solution)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

2 R 3 N

5 N

32*

0

0

1

0

1

5

5 R

5 R

4 R

5 R

56

0

1

0

0

1

5

4 N 1 R

3 N 1 R

5 N

3 R 2 N

100

5

4

4

4

17

85

A/I

1 R

1 R

1 R


*      Increased number of sub lethal effects observed when compared to the control group, considered not to have had an impact on the outcome of the test.

R1– R4= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced movement

P = Pale

A/I = All daphnia immobilized

Positive control:

Time Point

(Hours)

EC50
(mg/L)

95% Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

24

1.3

1.0

-

1.8

1.0

1.8

48

1.2

1.1

-

1.3

0.56

1.0

Validity criteria fulfilled:
yes
Remarks:
None of the control daphnids showed immobilization or other signs of disease or stress and the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
Conclusions:
The 48h-EC50 value to Daphnia magna is 0.43 mg/L based on geometric mean measured test concentrations.
Executive summary:

A study was performed to assess the acute toxicity of the test substance to Daphnia magna. The study was conducted in compliance with OECD TG 202 and GLP. The test substance was a poorly soluble pure compound and as such prepared as a saturated solution. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56, 100 % v/v saturated solution for 48 hours under semi-static test conditions. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 0.051 to 0.66 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.039 and 0.60 mg/L and hence the results were calculated based on the geometric mean measured test concentrations. The range tested based on geometric mean measured test concentrations was 0.052, 0.084, 0.16, 0.27 and 0.62 mg/L. The 48-hour EC50 value to Daphnia magna was 0.43 mg/L based on the geometric mean measured test concentrations.

Description of key information

The 48-hour EC50 value to Daphnia magna (OECD TG 202): 0.43 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.43 mg/L

Additional information

A study was performed to assess the acute toxicity of the test substance to Daphnia magna. The study was conducted in compliance with OECD TG 202 and GLP. The test substance was a poorly soluble pure compound and as such prepared as a saturated solution. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56, 100 % v/v saturated solution for 48 hours under semi-static test conditions. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 0.051 to 0.66 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 0.039 and 0.60 mg/L and hence the results were calculated based on the geometric mean measured test concentrations. The range tested based on geometric mean measured test concentrations was 0.052, 0.084, 0.16, 0.27 and 0.62 mg/L. The 48-hour EC50 value to Daphnia magna was 0.43 mg/L based on the geometric mean measured test concentrations.