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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:
2017-08-10 to 2017-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted April 13 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Batch no.: 201701151
- Purity: 99.235% (area-% (HPLC))
- Expiry date: 01 December 2018
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 120.0 mg/L (nominal, limit concentration)
- Sampling method: 10 mL sample was extracted once with 10 mL hexane (liquid-liquid extraction). A portion of hexane was removed for analysis, and diluted into calibration range with hexane. Matrix matched calibration standards were similarly prepared by fortification of 10 mL control Elendt M4 medium with 1,1-diethoxyethane and extracting with 10 mL hexane. Quantification was by gas chromatography with mass spectrometry detection (GC-MS).
- Sample storage conditions before analysis: frozen
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
A 120 mg/L test solution was prepared by adding 36.70 mg of test substance to 305 mL of Elendt M4 medium and the test solution was stirred for 1 hour at room temperature in the dark test media code = TM1). After the 1 hour stir period, the test solution was collected and checked for the tyndall effect using a turbidity meter. The turbidity measurement for TM1 and the control was 0.254 NTU (Nephelometric Turbidity Units) and 0.248 NTU, respectively, indicating that similar particulate was present in both samples and that TM1 was therefore in solution.

The negative control treatment was prepared with Elendt M4 medium only using the same stirring procedure as used for TM1.

- Controls: negative and positive comtrol
- Evidence of undissolved material: no; the test preparations were observed to be colourless solutions throughout the duration of the test. No precipitation of test substance was observed during the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: MicroBioTests Inc, Belgium
- Age of parental stock: juvenile Daphnia magna (less than 24 hours old)
- Feeding during test: no
- Food type: concentrated suspension of Chlorella vulgaris
- Frequency: fed daily during culture

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Cultures were usually discarded at or before day 35. Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before a test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
No
Hardness:
250 mg CaCO3/L
Test temperature:
Continuous Water Temperature Range: 19.1 – 20.3°C
pH:
Test item: 7.86 - 8.01
Control: 7.78 - 8.14
Dissolved oxygen:
Test item: 116% (10.61 mg/L) in fresh medium, 104% (9.69 mg/L) in old medium (after 48 hours)
Control: 115% (10.49 mg/L) in fresh medium, 110 % (10.27 mg/L) in old medium (after 48 hours)
Salinity:
Not applicable
Conductivity:
0 hour (new):
Test item: 625 µS/cm
Control: 620 µS/cm

48 hours:
Not available
Nominal and measured concentrations:
Nominal: 0 (negative control), 120 mg/L
Measured (mean): 104 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: jars
- Type: closed
- Material, size, headspace, fill volume: 50 mL capacity glass jars with screw cap lids, completely filled and sealed to prevent the loss of test substance
- 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: Elendt M4 Media
- Particulate matter: no
- Culture medium different from test medium: no
- Intervals of water quality measurement: all water quality and environmental conditions were measured using calibrated meters; hardness and alkalinity of the Elendt M4 Media used for the test was measured before use on the test.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light : 8 hour dark cycle
- Light intensity: not specified, fluorescent lighting

EFFECT PARAMETERS MEASURED
The measured effect parameter was the immobility of daphnids after 48 hours of exposure. An individual was considered immobile if, when the contents of the test vessel were briefly agitated, it did not swim during a 15-second period of observation. In addition, Daphnia magna submerged in the body of the test media and those that were held at the surface of the test media were also recorded.


RANGE-FINDING STUDY
- Test concentrations: 0 (negative control), 0.12, 1.2, 12 and 120 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes. The results of the range-finding test suggested that the 48-hour EC50 value would be > 120 mg/L (based on nominal test substance concentration).
Reference substance (positive control):
yes
Remarks:
potassium dichromate. Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 104 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 104 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 104 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 104 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 104 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
> 104 mg/L
Nominal / measured:
meas. (arithm. mean)
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 (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no. A non-GLP analytical solubility/stability trial was conducted between 16 August 2017 and 18 August 2017 at a limit test concentration of 120 mg/L.

The validity criteria for control immobility (≤ 10 %) and dissolved oxygen (≥ 3 mg/L) were both satisfied. Therefore, the test is considered as valid.
Results with reference substance (positive control):
Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity. Results of the last test (2016-09-05):

24-h EC50 = 1.37 mg/L
48-h EC50 = 0.626 mg/L

The 24-h EC50 result is within the expected range stated in OECD Guideline 202 (0.6 - 2.1 mg/L)
Reported statistics and error estimates:
No statistical analysis was performed on this test, as it was performed as a limit test and there was no test substance effect observed.

Table 1: Analysis of the test item in the test media during the definitive test

Nominal concentration (mg/L)

Measured concentration
(mg/L)

Mean neasured concentration
(mg/L)

0 hours
(new media)

% of nominal

48 Hour
(Old media)

% of nominal

Control

-

NC

-

NC

-

120

110

91.7

98.0

81.7

104

-      Measured concentration below limit of quantification (LOQ = 0.1 mg/L)

NC   Not Calculated

Table 2: Immobilisation of Daphnia magna after 24 hours exposure during the Definitive test

Nominal concentration
(mg/L)

Mean measured concentration
(mg/L)

Number of
Daphnia magna
exposed

MobileDaphnia magna

ImmobileDaphnia magna

Submerged

Surface

Submerged

Surface

Control

Control

20

20

-

-

-

120

104

20

20

-

-

-

 

-      Not applicable

Table 3: Immobilisation of Daphnia magna after 48 hours exposure during the Definitive test

Nominal Concentration
(mg/L)

Mean measured concentration
(mg/L)

Number of
Daphnia magna
exposed

Mobile Daphnia magna

Immobile Daphnia magna

Submerged

Surface

Submerged

Surface

Control

Control

20

20

-

-

-

120

104

20

20

-

-

-

-      Not applicable

Validity criteria fulfilled:
yes

Description of key information

The 48-h EC50 of the test item was determined to be > 120 mg/L (nominal).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
120 mg/L

Additional information

To determine the 48-hour acute toxicity of the test substance against the mobility of the freshwater planktonic crustacean,Daphnia magna., an Acute Immobilisation Test according to OECD Guideline 202 was conducted in compliance with GLP criteria (BASF SE, 2017). Based on the results of a range finding test, the main test was performed at the limit concentration of 120 mg/L (nominal). A negative control was run in parallel. Test vessels (ca. 50 mL capacity glass jars with screw caps) were prepared containing ca. 50 mL of the appropriate test or control medium. The test concentration was prepared by stirring the test substance in Elendt Media for 1 hour in the dark. At the start of the exposure, five juvenile daphnids were added to each test vessel. The daphnids in each test vessel were observed at 24 and 48 hours. The daphnids were not fed during the test. Analysis of the 120 mg/L test concentration at 0 hours showed a measured concentration of 110 mg/L. Analysis of the corresponding old media sample at 48 hours showed a measured concentration of 98.0 mg/L. Given that the mean measured concentration was within ± 20 % of the nominal concentration (86.7 %), it was considered justifiable to base the results on nominal concentrations as recommended in the OECD 202 guideline. Nevertheless, the results have also been based on mean measured concentrations. The 48-h EC50 was determined to be > 120 mg/L (nominal) and > 104 mg/L (measured, arithmetic mean), respectively. The 48-h NOEC was determined to be ≥ 120 mg/L (nominal) and ≥ 104 mg/L (measured, arithmetic mean), respectively. No immobilisation was observed in the control after 24 hours and 48 hours. The validity criteria for control immobility (≤ 10 %) and dissolved oxygen (≥ 3 mg/L) were both satisfied. The test is therefore considered to be valid.