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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
other: EEC Guideline XI/681/86, Draft 4: "Prolonged toxicity study with Daphnia magna: Effects on reproduction"
equivalent or similar to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
Principles of method if other than guideline:
21d semistatic long-term toxicity test assessing the effect of sublethal concentrations of the test substance on reproductive output of Daphnia magna
GLP compliance:
Specific details on test material used for the study:
- Batch No.: 31-9412
- State of aggregation: powder
- Purity of active ingredient: > 89%
- Impurities / secondary components: see characterisation from ZAX-report No. 96L00703
Analytical monitoring:
Details on sampling:
Samples for analysis were taken in the 1st, the 2nd and the 3rd week of the test. For each concentration the freshly prepared test solution (unstocked) and the corresponding 48 h or 72 h old test solution (stocked with daphnids) were analyzed.
Details on test solutions:
At the beginning of the test and before changing the test solutions, a stock solution of the test substance was freshly prepared. A defined amount of the substance was weighed out and suspended directly in the test medium by stirring (about 10 min at 22 ± 1 °C). The nominal concentrations of the stock solutions were 100 mg/L. By dilution of the stock solutions with test medium, the nominal test concentrations were prepared.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Source: inhouse culture originally obtained from Institut National de Recherche Chimique Appliquée, France, 1978
- Age at study initiation (mean and range, SD): < 24h
- Feeding during test
- Food type: living green algae (D. subspicatus)
- Amount: days 0-1: 0.22 mg COD; days 2-3: 0.25 mg COD; days 4-5: 0.35 mg COD; days 6-7: 0.43 mg COD, days 8 -21: 0.75 mg COD
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
2.20 - 3.20 mmol/L
Test temperature:
19.9 - 21.7 °C
Dissolved oxygen:
8.0 - 9.6 mg/L
550 - 650 µS/cm
Nominal and measured concentrations:
Nominal: 100; 50.0; 25.0; 12.5; 6.25; 3.13 and 1.56 mg/L
Measured: > 80% nominal
Details on test conditions:
- Test vessel: 100 mL beaker containing 50 mL test solution
- Renewal rate of test solution (frequency/flow rate): three times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

- Source/preparation of dilution water: Elendt M4 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was measured continuously in a separat beaker.DO and pH were recorded at every renewa lin the old and new test solution (in one beaker per concentration).

- Photoperiod: 16h light: 8h dark
- Light intensity: 5-6 µE/(m2*s)

EFFECT PARAMETERS MEASURED: Reproductive output and mortality was recorded daily theroughout the test.
Reference substance (positive control):
Key result
21 d
Dose descriptor:
Effect conc.:
25 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
50 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: NOEC = 2.5 µg/L
Reported statistics and error estimates:
For the statistical evaluation of the LOEC and NOEC Duncan's new multiple range test was used.

Analytically found relative minimum and maximum concentrations of the test substance in the test solutions at different times of the test (with and without daphnids). The values are given in percent of the nominal concentrations:

 Time [h]  stocked with daphnids  Min [%]  Max [% ]
 0  no  80.8  166.7
 48  no  92.8  166.7
 48  yes  95.2  173.1

M4 -water caused a background value of about 1 mg/L of the test material. Regarding to this and to the precision of the analytical method, most concentrations found corresponded to the expected values.

Validity criteria
In the control:
- the mortality of parent animals up to the end of the test was <= 10 %.
- the mean number of living young per parent animal that survived the test was >= 60.
- the coefficient of variation for the mean number of living young per parent animal that survived the test was <= 25 %.
- The NOEC of the control substance 3,4-dichloroaniline was 2.5 µg/L, the LOEC was 10 µg/L and LC0 was > 10 µg/L.
The validity criteria were fulfilled by the test.

In the control the first young were observed at day 7. In the highest concentration tested, at which the daphnids produced young (100 mg/L), the first young were observed at day 13.

Validity criteria fulfilled:
The test substance is not harmful to aquatic invertebrates at chronic exposure.

Description of key information

NOEC(21d) = 25 mg/L for reproduction of Daphnia magna (OECD 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
25 mg/L

Additional information

One study is available investigating the long-term toxicity of Disodium dihydrogen ethylenediaminetetraacetate (CAS No. 139-33-3) is available. The key study was performed according to EEC Guideline XI/681/86, Draft 4 : "Prolonged toxicity study with Daphnia magna: Effects on reproduction", which is equivalent to OECD guideline 211 (BASF 1998). The semi-static study was conducted under GLP conditions using Daphnia magna as test organism (BASF 1998). Concentrations between 1.56 and 100 mg/L were tested. and analytically verified (> 80% of nominal). The 21d-NOEC value for reproductive output is determined to be 25 mg/L. Being the lowest NOEC value of all three trophic levels, this value is used for the derivation of PNEC freshwater and marine water.