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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 - 28 Sep 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Germany

Test material

Constituent 1
Details on test material:
- Name of test material: reaction mass of potassium sodium (2R,3R)-2-hydroxy-3-(phosphonatooxy)butanedioate and potassium sodium (2S,3S)-2-hydroxy-3-(phosphonatooxy)butanedioate
- Physical state: white to slightly yellow powder of crystalline appearance
- Lot/batch No.: 010-2065
- Expiration date of the lot/batch: 16 Sep 2019
- Chemical stability: insensitive to exposure to light, considered to be stable in alkaline and neutral solutions (6 < pH <1 2)
- Storage condition of test material: storage at < 40 °C in a tightly closed container under dry conditions (substance very hygroscopic)

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: at test start samples of control media and of each test concentration were taken from the test solution preparation for analytical measurement of test item concentration just before distributing it to the four replicates. Additionally, a sample of the aged test solution was taken from each vessel and pooled per concentration.
- Sample storage conditions before analysis: Samples were acidified for storage. Potassium concentrations were determined within one week after sampling. For more details see section `Details on analytical methods´

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solution (100 mg test item/L) was prepared in dilution water. The individual test solutions (freshly prepared before test start) were prepared by dilution of the stock solution with dilution water, sampled for later chemical analysis and distributed to the test beakers
- Controls: only dilution water used

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Source: German Federal Environment Agency, Institut für Wasser-, Boden und Lufthygiene. Specimens used in the test were bred in the laboratory of the Fraunhofer IME
- Age at study initiation (mean and range, SD): 4 - 24 h
- Method of breeding: adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for 1 week. The water was changed once per week. Newborn D. magna were separated by sieving, the first generation was discarded. Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.
- Feeding during breeding: daily with an algal suspension (Desmodesmus subspicatus in the log-phase) and LiquizellR (HOBBY).
- Feeding during test: no

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

1.1 - 1.3 mmol/L
Test temperature:
20.2 - 20.4 °C
8.1 – 8.4
Dissolved oxygen:
8.6 - 9.1 mg/L
Nominal and measured concentrations:
Nominal concentrations: control, 19.8, 29.6, 44.4, 66.7, 100 mg/L test item (corresponding to 6.12, 9.19, 13.8, 20.7, 31 mg potassium/L, nominal)
Measured concentrations at test initiation: 0.8996, 6.20, 9.30, 13.9, 21.0, 30.9 mg potassium/L (after background correction)
Details on test conditions:
- Test vessel: glass beakers (50 mL nominal volume). Beakers were covered with glass panes to prevent from evaporation but also permit gaseous exchange between the medium and the atmosphere and access of light.
- Fill volume: 50 mL
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4 replicates with 5 specimens per replicate
- No. of vessels per control (replicates): 4 replicates with 5 specimens per replicate

- Source/preparation of dilution water: purified (filtrated with activated charcoal, passed through a limestone column, and aerated) drinking water was used as holding- and dilution water. To avoid copper contamination, plastic water pipes are used for the testing facilities.
- Water chemistry data recorded regularly in the testing facility: pH, conductivity, dissolved oxygen content, content of nitrate, nitrite, ammonium, phosphate, calcium, magnesium, total hardness, alkalinity, DOC content (or NPOC, as appropriate), content of metals (copper, iron, manganese and zinc)
- Hardness: in spite of the passage through a lime-stone column, hardness was below the recommended (not required) range (140 - 250 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Historical data showed values of 70 – 120 mg/L as CaCO3. However, according to the guideline lower hardness may be also appropriate for other Daphnia species. Historical results from reference tests at the testing laboratory indicate that test conditions are also suitable for the Daphnia magna strain.
- Chlorine: <0.002 - 0.02 mg/L
- Alkalinity: 1.8 – 2.4 mmol/L
- Ca/mg ratio: 1.0 - 1.2/ 0.1 mmol/L
- Conductivity: 228 – 227 μS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: oxygen concentration, pH and temperature of the test solutions were checked at test start and end

- Photoperiod: 16/8 h
- Light intensity: 904 – 963 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : the numbers of dead animals were visually determined daily and dead specimens (if occurred) were removed

- Range finding study: yes, non GLP study, no results in the report
Reference substance (positive control):

Results and discussion

Effect concentrations
48 h
Dose descriptor:
Effect conc.:
66 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL: 56.1 - 80.9 mg/L
Details on results:
- Behavioural abnormalities: no
- Other biological observations: no

Results with reference substance (positive control):
- EC50: 1.32 mg/L
Reported statistics and error estimates:
Biological effect values were calculated by Probit-analysis assuming log-normal distribution of the values. All statistical calculations were performed
using the computer programme ToxRat®.

Any other information on results incl. tables

Table 1: % of immobilisation after 24 h and 48 h. Mean values. TI = Test item

Incubation  Control  19.8 mg TI/L  29.6 mg TI/L  44.4 mg TI/L  66.7 mg TI/L  100 mg TI/L
24 h  0 0 0 10 50* 65*
48 h 0 0 5 25 55* 75

* significant difference when compared with control

Table 2: NOEC and ECx values for immobilisation at test end [mg TI/L]. Effect concentrations given as nominal values.

Mortality mg TI/L
NOEC  44.4
LOEC  66.7
EC10 (95% CL)  34.0 (23.8 - 41.5)
EC20 (95% CL)  42.7 (33.1 - 50.5)

Table 3: Measured concentrations of potassium (K) in the test item (TI).

Nominal concentration Measured concentration Measured concentration (minus background)
mg TI/L  mg K/L 0 h mg K/L  48 h aged mg K/L  0 h mg K/L 0 h % nominal  48 h aged mg K/L  48 h aged % nominal % initial
Control  - 0.8996 0.9399
19.8 6.12 7.1 7.19 6.2 101 6.25 102 101
29.6 9.19 10.2 10.8 9.3 101 9.82 107 106
44.4 13.8 14.8 15.3 13.9 101 14.4 104 104
66.7 20.7 21.9 22.6 21 102 21.7 105 103
100 31 31.8 32.6 30.9 99.6 31.6 102 102

The Daphnia magna, acute immobilisation test fulfils the validity criteria of the guideline:

·        With 0% the percent mortality observed in the controls over the test period is ≤ 10%.

·        The dissolved oxygen concentration at the end of the aging period (48 h) was ≥ 3 mg/L in control and test vessels.

Applicant's summary and conclusion