Reaction mass of potassium sodium (2R,3R)-2-hydroxy-3-(phosphonatooxy)butanedioate and potassium sodium (2S,3S)-2-hydroxy-3-(phosphonatooxy)butanedioate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 - 28 Sep 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Germany

Test material

Test material information

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:

yes

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

Vehicle:

no

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:

TEST ORGANISM
- 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

1.1 - 1.3 mmol/L

Test temperature:

20.2 - 20.4 °C

pH:

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 SYSTEM
- 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

TEST MEDIUM / WATER PARAMETERS
- 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

OTHER TEST CONDITIONS
- 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

TEST CONCENTRATIONS
- Range finding study: yes, non GLP study, no results in the report

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

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.