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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2 Oct 2012
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: The concentration of the test substance was determined in one representative interval per week in freshly prepared test solution (test vessels without daphnids) and in the 24 hour old test solutions before renewal (combined sample from test vessels with daphnids). Aged biotic replicates were not sampled since the presence of food algae interferes with the analytical measurement.
- Sample storage conditions before analysis: stored in a freezer.
Details on test solutions:
- Method: The test substance is a mixture that is poorly soluble in water. Therefore the test solutions were prepared following general guidance provided in OECD 23 in order to achieve a water accommodated fraction (WAF) of the test substance using a liquid-liquid saturator technique. Generally, each test solution was prepared separately by directly
adding test substance to test medium. A defined volume of test media was placed into glass aspirator bottle (2-L) with a bottom side-outlet attached to a stopcock. The necessary volume of the test substance (according to the table below) was calculated from the density. Prior to use the test substance was homogenized by shaking the container. Then the test substance was pipetted carefully on the water surface. The bottle was closed and the solution was stirred on a magnetic plate for approximately 1 day. The stirring was slow enough (approx. 100 rpm) so that no vortex formed, preventing foaming or emulsification. Stirring was stopped for 1 hour and any undissolved material was allowed to separate.The required volume of the WAF (test solution) was drawn off for testing via the bottom port of each bottle.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were visibly colorless and clear throughout each renewal period.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Strain/clone: 1820
- Source: in-house culture originally obtained from Institut National de recherche Chimique Appliquee, France, in 1978
- Feeding during test
- Food type: live green algae (D. subspicatus)
- Amount: < 134 µL of a concentrate of 1490 - 1760 mg TOC/L to reach the following amount of food: 0-3d: 0.1 mg TOC/parent animal and day; 4-5d: 0.12 mg TOC/parent animal and day; 6-7d: 0.14 mg TOC/parent animal and day; 8-21d: 0.2 mg TOC/parent animal and day
- Frequency: daily

- Acclimation period: The daphnids are cultured under the identical conditions as the test including test media and diet.
- Acclimation conditions (same as test or not): same
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
2.45 - 2.47 mmol/L
Test temperature:
20 °C
7.8 - 8.2
Dissolved oxygen:
8.5 - 9.2 mg/L
Nominal and measured concentrations:
Nominal: 1.25, 2.5, 5.0, 10 and 20 mg/L loading rate WAFs
Details on test conditions:
- Test vessel: 100 mL glass beakers with 50 mL test solution
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.02 animals/mL

- Source/preparation of dilution water: synthetic fresh water (Elendt M4)
- Total organic carbon: < 2 mg/L
- Alkalinity: 0.8 - 1.00 mmol/L
- Ca/mg ratio: 4:1
- Conductivity: 550 - 650 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in replicate 1 of each test group for one interval per week. In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels. Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.

- Photoperiod: 16h light: 8h dark
- Light intensity: 658 - 728 lux at wave length of 400 - 750 nm

EFFECT PARAMETERS MEASURED: Parent mortality, reproductive output and time to first brood were recorded daily.
Reference substance (positive control):
sodium chloride
Key result
21 d
Dose descriptor:
Effect conc.:
>= 20 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
> 20 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
Details on results:
- Behavioural abnormalities: none
- Observations on body length and weight: none
- Other biological observations: none
- Mortality of control: One daphnid died in the control group (10% mortality)
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 48h-EC50 = 4.48 mg/L
- Other: The EC50(48 h) of the reference substance sodium chloride (NaCl) was 4.48 g/L (experiment date: 20 Oct 2015, project number: 50E0789/12E046. This result is within the range of 3.88–7.22 g/L, which represents ±2 standard deviations from the published EC50(48 h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Test substance analytics

The measured TOC in each treatment increased proportionally to the loading rate as expected for WAF preparations. The higher TOC is likely due to soluble organic components originating from the test substance. TOC values from the start and end of each exposure interval were similar and confirm that exposure was consistent over the entire experiment. The TOC values confirm qualitatively that the daphnids in each WAF were exposed to soluble organic components from the test substance. According to guidance in OECD 23, for tests with chemicals that cannot be quantified by analytical methods at theconcentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or in the case of water accommodated fractions on the loading rate.

Validity criteria fulfilled:
Kerocom FM 38 is not harmful to aquatic invertebrates based on chronic exposure.

Description of key information

NOELr (21d) >= 20 mg/L (nominal, loading rate WAF) for reproductive output of Daphnia magna (OECD 211)

Key value for chemical safety assessment

Additional information

On study investigating the long-term toxicity of Fatty acids, C8-18 and C18-unsatd., reaction products with diethanolamine and propylene oxide (CAS 1000817-22-0) to aquatic invertebrates is available. The study was performed according to GLP and OECD guideline 211 using Daphnia magna as test organism (BASF 2016). Loading rate WAFs between 1.25 and 20 mg/L were tested. The test was performed under semi-stic conditions. No inhibition of reproduction was observed in any treatment compared to the control. Thus, the 21d-NOELr is determined to be >= 20 mg/L based on nominal concentrations.