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Ecotoxicological information

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
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
May 2008
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
October 2012
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: At the beginning, after 24 hours and on day 7, all test and blank control solutions were measured. Test item was not detectable in the test solutions, neither in test solutions with nor without daphnia and algae. Thus, the number of analytical measurements was reduced. From day 14 until the end of the test, the test item concnetration was measured only in the control and the highest concentrated treatment (nominal 4.6 mg/L) in the old (with and without daphnia) and new solutions.
- Sampling method: Samples were diluted with an equal volume methanol before measurement (500 µL sample were mixed with 500 µL methanol in a HPLC vial).
- Sample storage conditions before analysis: Samples were analysed without prior storage.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock WAF containing the nominal load 4.6 mg/L was prepared by weighing the nominal load, adding the corresponding amount of dilution water and shaking vigorously for 23-24 hours. The resulting solution was filtrated through 0.45 μm filters. The lower treatments (2.3 / 1.1 / 0.11 / 0.011 mg/L) were prepared by dilution of the WAF with dilution water.
The highest test concentration (nominal 4.6 mg/L) was tested in the absence and presence of 10 mg/L of sodium salt of humic acid.
- Controls: dilution water with and without 10 mg/L of sodium salt of humic acid.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flee
- Strain/clone: STRAUSS
- Source: in-house culture originally obtained from the Umweltbundesamt (Federal Environment Agency) in Berlin, Germany in 2007
- Feeding during test
- Food type: green algae (Desmodesmus subspicatus)
- Amount: 0.20 mg organic carbon/animal/day
- Frequency: daily

- Acclimation period: none
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: green algae (Desmodesmus subspicatus)

Similar to the method described in EN ISO 6341. 22.5 hours before the start of the test, the adult animals were separated from the young. 21.5 hours later, the adults were caught with the help of a glass tube, and the newborn daphnia, aged between 0 and 20 hours, were sieved from the medium and immediately placed into a 250 ml-beaker containing dilution water. After a settling-in period of 30 minutes, animals which showed no apparent damage were used for the test.
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
267 mg CaCO3/L
Test temperature:
Dissolved oxygen:
8.4-9.9 mg O2/L
Nominal and measured concentrations:
nominal: 4.6, 2.3, 1.1, 0.11 and 0.011 mg/L (nominal concentrations were based on the results from a first experiment, where a NOEC could not be determined (< 4.6 mg/L loading rate)
measured: < LOD
Details on test conditions:
- Test vessel: glass beakers, nominal volume 100 mL
- Aeration: none
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 80 mL/animal

- Source/preparation of dilution water: deionised water with an enrichment of certail minerals was used in the test. The minerals which were added comply with the composition of Elendt- M4 medium (as stated in the OECD-Guideline)
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: no

- Adjustment of pH: no
- Photoperiod: 16/8 hours
-Lighting: 1000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The test vessels were inspected for offspring daily. The newborn daphnids were counted and removed from the test vessel.

- Spacing factor for test concentrations: 2 and 10
- Range finding study
- Results used to determine the conditions for the definitive study (based on loading rates):
short-term toxicity test to Daphnia: 48-h EL50 15 mg/L (tested concentrations: nominal 4.27, 9.39, 20.7, 45.5, and 100 mg/L as WAF)
first long-term toxicity test to Daphnia: NOEC < 4.5 mg/L (tested concentrations: nominal 4.6, 10, 22, 46, and 100 mg/Las WAF)

Reference substance (positive control):
not required
Key result
21 d
Dose descriptor:
Effect conc.:
>= 2.3 - < 4.6 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: without humic acid
21 d
Dose descriptor:
Effect conc.:
>= 4.6 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: with humic acid (10 mg/L of sodium salt of humic acid)
21 d
Dose descriptor:
Effect conc.:
4.6 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: without humic acid
Details on results:
- Behavioural abnormalities: none
- Other biological observations: in replicates with humic acid (10 mg/L sodium salt of humic acid with or without nominal 4.6 mg/L test material) reproduction was inhibited (see Table 1 in "Any other information on results incl. tables")
- Mortality of control: blank control: 10% (1 out of 10); blank control with humic acid: 0%
- Other adverse effects control: none
- Abnormal responses: none
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological results was accomplished using the software Tox-Rat® Professional, version 3.2.1.

A clear decrease in the reproduction of the daphnia was observed in the control replicates with 10 mg/L sodium salt of humic acid (46%; see Table 1).

A slight but statistical significant decrease of 23% in the reproduction was observed only at the highest concentration tested (nominal 4.6 mg/L) in the absence of humic acid (see Table 1).

No difference in the reproduction was observed at the highest concentration tested in the presence of humic acid compared to the blank control with humic acid (HA) (see Table 1)

Table 1 Fecundity


Total Offspring

Surviving Parent Animals

Mean Offspring per Surviving Parent Animal

Inhibition of Reproduction in %

Blank Control



107 0
Blank Control HA 576 10 58 46
 0.011 mg/L 938 10 94 13
 0.11 mg/L 1017 10  102 5
 1.1 mg/L 982 10 98 8
 2.3 mg/L 769 8 96 10
 4.6 mg/L 823 10 82 23
 4.6 mg/L HA 490 9 54 49

The influence of the test material was additionally tested in the presence of humic acid to mimic environmental conditions. Although humic acid had a clear detrimental effect on the reproduction of the daphnia, the toxic effect of the test material itself as observed at the nominal concentration of 4.6 mg/L was no longer observed in the presence of humic acid.

Validity criteria fulfilled:
mortality of parent animals in the blank control: ≤ 20%; mean surviving offspring in the blank control: ≥ 60
The test material did not exhibit toxic effects on the reproduction of Daphnia up to its water solubility.

Description of key information

21-d NOEC (based on loading rates) 2.3 mg/L for Daphnia magna (OECD 211, EU C.20)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.3 mg/L

Additional information

The results indicate that the test material is not toxic to the reproduction of Daphnia up to the limits of its water solubility and thus a PNEC cannot be determined.