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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:
06/2016 to 08/2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Source and lot/batch No.of test material: 15112601
- Expiration date of the lot/batch: Nov. 2020
- Purity test date: Nov. 2015

- Storage condition of test material: Room Temperature (20 ± 5°C); Keep away from humidity
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: <0.1g/L;
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not known

- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final preparation of a solid: applied as saturated solution
Analytical monitoring:
Details on sampling:
- Concentrations:
4.6 to 100 mg/L nominal
1.3 to 35.1 mg/L measured
- Sampling method: HPLC
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A saturated solution of the test item was prepared for the test. This was done by weighing
the nominal load 100 mg/L (exact loads are given below), adding the corresponding
amount of dilution water and shaking vigorously for 24 hours. The resulting solution was
filtrated through 0.45 μm filters (Nylon). The lower treatments were prepared by diluting the
saturated solution with dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Strain/clone: STRAUS Berlin

- Source: LAUS GmbH
- Age of parental stock (mean and range, SD): 0 to 24 hours
- Feeding during test
- Food type: green algae (Desmodesmus subspicatus)
0.15 - 0.20 mg org. carbon/animal/day, the amount of
carbon is based on photometric measurement (linear correlation
between absorption at 440 nm and measured
DOC concentration).
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
267 to 356 mg CaCO3/l
Test temperature:
19.4 - 22.2°C
7.4 to 8.5
Dissolved oxygen:
7.7 to 10.8 mg/l
Nominal and measured concentrations:
4.6 to 100 mg/L nominal
1.3 to 35.1 mg/L measured
Details on test conditions:
- Test vessel: Glass beakers (nominal volume 100 mL)
- Type (delete if not applicable): open
- Aeration: passive
- 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
- No. of vessels per vehicle control (replicates): not applicable

- Source/preparation of dilution water:
Deionised water with an enrichment of certain minerals was used in the test. The minerals
which are added comply with the composition M4 (full composition see annex 5, page 42)
derived by ELENDT as stated in the OECD-Guideline.

- Adjustment of pH: not required
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: 1000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
reproduction rate

- Spacing factor for test concentrations: ca. 2.2
- Test concentrations:
4.6 to 100 mg/L nominal
1.3 to 35.1 mg/L measured
Key result
21 d
Dose descriptor:
Effect conc.:
7.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Key result
21 d
Dose descriptor:
Effect conc.:
16.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
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
The number of offspring in treatment 10 mg/L (measured concentration 3.3 mg/L) was
higher than in the control and in this treatment no mortality was observed. Therefore only
treatment 22 mg/L (measured concentration 7.7 mg/L) was used for statistical evaluation.
The details of calculation are stated in Annex 4: Statistical calculation using ToxRat® Professional
3.2.1. Biological results were stated using two significant digits.


SD = standard deviation; RSD = relative standard deviation = coefficient of variation M = mortality of parent animal

Replicate Blank control 4.6 mg/L 10 mg/L 22 mg/L 46 mg/L 100 mg/L
1 M 115 125 M 0 M
2 119 117 90 106 M M
3 125 78 116 M 0 M
4 138 M 87 119 M M
5 123 130 146 58 0 M
6 84 M 108 M M M
7 114 115 135 84 M M
8 116 44 101 105 0 M
9 70 96 103 M M M
10 M 127 121 108 M M
Mean 111.1 102.8 113.2 96.7 0 --
SD 22.6 29.1 19.0 22.1 -- --
RSD 20.3 28.4 16.8 22.8 -- --

Summary of Fecundity

Treatment Total Offspring Surviving Parent Animals Mean Offspring per Surviving Parent Animal Inhibition of Reproduction in %
Blank Control 889 8 111 0
4.6 mg/L 822 8 103 8
10 mg/L 1132 10 113 -2
22 mg/L 580 6 97 13
46 mg/L 0 4 0 100
100 mg/L 0 0 0 100
Validity criteria fulfilled:
In the OECD 211 study, the NOEC and the LOEC were determined to be 7.7 mg/L and 16.5 mg/L, respectively.
Executive summary:

One valid experiment was performed. The study was performed using 5 concentrations ranging from 4.6 to 100 mg/L nominal concentration. 10 daphnia each were exposed to the test item and the blank controls for 21 days. The offspring of the animals was counted each working day. Reproduction in the treatments 4.6 mg/L – 22 mg/L did not statistically deviate from reproduction in the blank control whereas in the treatments 46 mg/L and 100 mg/L no reproduction was observed. In the treatment 100 mg/L, until day 9 all daphnia died. In treatment 46 mg/L only 4 daphnia survived until the end of the test. The number of offspring in treatment 10 mg/L was higher than in the blank control and in this treatment no mortality was observed. Therefore, only treatment 22 mg/L was used for statistical evaluation. The content of the test item in the test solutions was determined using HPLC determination. Because of the poor solubility of the test item, the correlation between nominal and measured concentration was weak. But the analysis of the test item concentrations in the treatments before and after exposition showed sufficient stability of the test item during exposition. Therefore, the determination of the results was based on the geometric means of the measured concentrations. The following results were determined for the test item BRUGGOLEN M12: NOEC = 7.7 mg/L LOEC = 16.5 mg/L

Description of key information

In the OECD 211 study, the NOEC and the LOEC were determined to be 7.7 mg/L and 16.5 mg/L, respectively.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
7.7 mg/L

Additional information