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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:
12 - 14 Apr 2016
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 (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and limit concentration (100 mg/L, nominal)
- Sampling method: At the start of the exposure (0 h), sampling was carried out after preparation of the limit concentration. At the end of the exposure (48 h), samples were taken from additional replicates, which were prepared with test media, but without daphnia. These additional replicates were incubated under test conditions until sampling.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until preparation, if necessary. Prepared samples were stored at room temperature in the autosampler until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the limit concentration, 100 mg/L of the test item was weighed out and was freshly prepared with dilution water before the start of the expsoure (0 h). The limit concentration was treated with ultrasound for 15 min at room temperature and stirred thereafter with a magnetic stirrer at approximately 1100 rpm for 15 rpm at room temperature.
- Differential loading: no
- Controls: yes, dilution water without test item was incubated under the same conditions as the test group
- Evidence of undissolved material: no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone 5
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Berlin, Germany
- Culture conditions: The daphnia were cultured at the test facility, in 2 - 3 L glass vessels, with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator with a 16 h photocycle (max. 20 µE * m-2 * s-1 light intensity).
- Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 - 180 mg CaCO3/L
- Age of daphnia at the start of the exposure: < 24 h
- Feeding during test: no

CULTURE CONDITIONS
- Food type: a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, obtained from Sammlung von Algenkulturen (SAG), Göttingen, Germany.
- Amount: > 1E+06 algae cells/mL
- Frequency: at least 5 times per week

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Juvenile daphnids were removed from the culture vessels at the latest 24 h before the start of the exposure and discarded. The juveniles born within the following period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
178 mg CaCO3/L (dilution water/Culture Medium Elendt M4)
Test temperature:
19 - 20 °C
pH:
At 0 h: 8.82 (control), 8.45 (limit concentration)
At 48 h: 7.72 - 7.80 (control), 7.45 - 7.72 (limit concentration)
Dissolved oxygen:
At 0 h: 9.87 mg/L (control), 8.48 mg/L (limit concentration)
At 48 h: 7.78 - 7.90 mg/L (control), 7.72 - 7.85 mg/L (limit concentration)
Conductivity:
490 µS/cm
Nominal and measured concentrations:
nominal: control, 100 mg/L
measured: < LOQ, 104 mg/L (0 h), 98.7 mg/L (48 h)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Type: loosely covered with watch glasses
- Material, size, headspace, fill volume: glass, 4 (ID) * 7 (H) cm; Volume = 50 mL; Fill volume test media = 20 mL; Headspace = 30 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Test medium: Culture Medium Elendt M4, according to OECD 202, Annex 3 (2004), with total water hardness adapted to 160 - 180 mg CaCO3/L (instead of 293.8 mg/L in the original recipe)
- Culture medium different from test medium: no
- Water quality paramaters & intervals of measurement: The pH-values, dissolved oxygen concentration, temperature, conductivity and total hardness of the dilution water were measured prior to the start of the exposure (0 h). At the start of the exposure (0 h), the water quality paramters of the freshly prepared media were measured in one additional replicate of the control and of the limit concentration without daphnia.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: max. 20 µE*m-2*s-1 (max. 1340 lx)

EFFECT PARAMETERS MEASURED:
- immobilisation: at 24 h and 48 h

RANGE-FINDING STUDY
- Range-finding study: yes, a non-GLP preliminary range finding test was conducted with two replicates per treatment group and control, each with ten daphnia.
- Test concentrations: 1, 10 and 100 mg test item/L (nominal)
- Results used to determine the conditions for the definitive study: 0% immobilisation after 48 h exposure to the highest concentration.
Reference substance (positive control):
yes
Remarks:
potassium dichromate p.a.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no
- Other biological observations: no
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: No, the limit concentration was visually clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.95 mg/L
Reported statistics and error estimates:
The EC50 values for the reference item was calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5.

The concentration of the test item was analytically verified via HPLC-DAD at the start of the expsure (0 h) and at the end of the exposure (48 h) in the limit concentration and in the control.

The measured concentration in the limit concentration was 104% of the nominal value at the start of the exposure (0 h) and 99% of the nominal value at the end of the exposure (48 h). The measured test item concentrations were within ± 20% of the nominal concentration. This indicates that the test item concentration was successfully maintained for the duration of the test.

Table 1: Measured Concentrations of the Test Item during the Definitive Test.

Sampling date:

2016-04-12

Start of the exposure (0 h)

2016-04-14

End of the exposure (48 h)

Start of analysis

2016-04-12

2016-04-14

Nominal test item concentration [mg/L]

 

Test item

 

Meas. conc. [mg/L]

%

Meas. conc. [mg/L]

%

100

104

104

98.7

99

control

< LOQ

< LOQ

Meas. conc.     = measured concentration of the test item, mean value of 2 injections, dilution factor taken into account

%                = percent of the nominal concentration of the test item

LOQ             = limit of quantification of the analytical method (2.00 mg test item/L)

Validity criteria fulfilled:
yes
Conclusions:
The study resulted in an EC50 (48 h) of > 100 mg/L (nominal) based on mobility of Daphnia magna (OECD 202).

Description of key information

EC50 (48 h) > 100 mg/L (nominal, OECD 202, Daphnia magna)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

There is one GLP guideline study available, which assessed the effects of the substance towards aquatic invertebrates according to the OECD guideline 202. In a static test Daphnia magna was exposed to a nominal limit concentration of 100 mg/L for 48 h. The limit concentration was previously established in a preliminary range-finding test. The concentration of the test item was analytically verified by HPLC-DAD analysis at the start of the exposure (0 h) and at the end of the exposure (48 h). The measured concentration was 104% of the nominal values at the start of the exposure (0 h) and 99% of the nominal value at the end of the exposure (48 h). The measured test item concentrations were within ± 20% of the nominal concentration. This indicates that the test item concentration was successfully maintained for the duration of the test and the effect concentrations can be based on the nominal values. After 48 h no effects on Daphnia magna were observed. The EC50 (48 h) value was > 100 mg/L (nominal).