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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
Study period:
06 Dec - 27 Dec 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
Only three replicates were used for the evaluation due to a technician error.
according to guideline
EU Method C.20 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance is a liquid which is poorly soluble in water. Therefore the test solutions were prepared following general guidance provided in OECD 23 in order to generate a water accommodated fraction (WAF) of the test substance in test medium. Each individual test replicate was prepared separately as a liquid-liquid saturator exposure system which comprised a glass beaker with a suspended organism chamber made of a glass tube with a stainless steel mesh bottom. The organism chambers were submerged in the beakers with test medium so that the top of the tube remained sufficiently above the surface of the test medium avoiding overflow from the surrounding surface layer in the beaker.
The test substance was pipetted on the water surface outside of the organism chamber. The test vessels were covered and shaken for about one day. Before the organisms were introduced into the organism chambers the water surface inside the organism chamber was checked for any undissolved test substance. The undissolved portion of the loaded test substance remained in contact with the test medium during the exposure. The organism chamber prevented the test organisms from any direct contact with the undissolved test substance, while allowing full exposure to all soluble components in the WAF.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Throughout the exposure period oily drops of undissolved test substance were observed on the water surface outside of the organism chambers, while no test substance was visible on the water surface within the organism chambers.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Age of parental stock (mean and range, SD): Daphnia brood stocks are kept in mass cultures consisting of approx. 20 – 30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests.
- Feeding during test: yes
- Food type: During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium.
- Amount: The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1583 mg, 1950 mg and 1984 mg TOC*/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤ 126 μL) of the concentrate.
- Frequency: daily

- Acclimation period: The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ± 1 °C), and diet.
- Acclimation conditions (same as test or not): same as test
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
2.52 - 2.58 mmol/L
Test temperature:
20 - 21 °C
7.6 - 8.1
Dissolved oxygen:
7.8 - 8.9 mg/L
Nominal and measured concentrations:
Nominal: control, 10 mg/L (as loading rate WAF)
Details on test conditions:
- Test vessel: glass beakers into which organisms chambers were placed (cylinders of transparent glass, 6 cm diameter, 10 cm high, with a bottom of stainless-steel grid of 0.2 mm x 0.2 mm)
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass; Size: 600 mL; Fill volume: 400 mL; Headspace: 200 mL
- Aeration: 24 h prior to use; none during exposure
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 0.025 animals/mL

- Source/preparation of dilution water: A synthetic fresh water (Elendt M4) is used as media for culture and test purposes
- Culture medium different from test medium: no

- Photoperiod: 16 h light / 8 h dark
- Light intensity: about 672 - 707 lux at a wave length of 400 - 750 nm

EFFECT PARAMETERS MEASURED: Mortality, reproduction and other sublethal effects were recorded daily for 21 days. Additional endpoints (i.e. growth, immobile young, time to first brood, or aborted eggs) were checked.
Reference substance (positive control):
sodium chloride
Key result
21 d
Dose descriptor:
Effect conc.:
>= 10 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
> 10 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
No significant mortality or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. The data were not sufficient to calculate ECx values for reproduction or mortality. However based on best professional judgment, a 21-day EC0 ≥ 10 mg/L is estimated.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (48 h): 4.05 g/L
- Other: The 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.

Analysis of the test substance preparations were not conducted for technical reasons. Since the water solubility of the test substance is < 0.05 mg/L, analyses of the test substance preparations would not provide useful data. No reliable method for analyses in the required concentration range could be developed.  

On day 4, one replicate in the treatment group was lost due to a technician error. So the evaluation was based on three replicates and a total of 30 parent animals. No other deviations were reported.

Table 1: Other biological observations among surviving parent animals in each concentration after 21 d exposure.

Test group [mg/L]

Growth [mean length, mm]

Mean immobile young (per surviving adult)

Mean days to first brood

Mean aborted eggs (per surviving adult)

0 (control)










Validity criteria fulfilled:

Description of key information

No effects up to the water solubility limit for reproductive output of Daphnia magna (OECD 211).

Key value for chemical safety assessment

Additional information

One GLP Guideline study according to OECD 211 is available investigating the toxicity of fatty acids, C8-10, C12-18-alkyl esters (CAS 95912-86-0) to aquatic invertebrates is available (Salinas 2013). Daphnia magna was exposed to a nominal loading rate of 10 mg/L (Water Accommodated Fraction). Chemical analysis of the test substance concentrations was not possible since no suitable analytical method could be developed to detect the test substance. No effects on reproduction of D. magna were observed up to the water solubility limit (< 0.05 mg/L) resulting in a NOELR (21 d) ≥ 10 mg/L. Based on this result it can be concluded that fatty acids, C8-10, C12-18-alkyl esters will not exhibit chronic effects on aquatic invertebrates up to the water solubility limit.