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Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July - 21 Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from all test item concentrations and from the control
- Sampling method: Duplicate samples were taken from the freshly prepared test media at test start. At test end the contents of test beakers of each treatment were poured together and duplicate samples were taken.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: Defined amounts of the test item were added directly to the test water for each test concentration and were carefully stirred for 24 hours in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period (1 hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration.
The highest test item concentration of 100 mg test item/L was prepared by mixing 100.48 mg test item into 1004.8 mL test water, for the test item concentration of 50 mg test item/L, 50.31 mg test item were mixed into 1006.2 mL test water, for the test item concentration of 25 mg test item/L, 25.20 mg test item were mixed into 1008 mL test water, for the test item concentration of 12.5 mg test item/L, 25.28 mg test item were mixed into 2022.24 mL test water and for the test item concentration of 6.25 mg test item/L, 32.24 mg test item were mixed into 5158.4 mL test water.
- Controls: Yes, test water without test item

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone 5
- Age at study initiation: 1.83 to 20.83 hours old
- Stage and instar at study initiation: The test organisms were not first brood progeny.
- Method of breeding: The daphnids were bred in the test facility under similar condition as in the test.
- Source: Test facility`s in-house laboratory culture
- Feeding during test : No

ACCLIMATION
- Acclimation conditions: Same as in the test
- Type and amount of food: The daphnids in the stock culture were fed with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of the test facility
- Feeding frequency: At least on all working days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

20.2 - 20.1 °C (Control)
20.0 - 20.3 °C (Test item concentrations)

pH:

7.8 - 8.0 (Control)
7.8 - 8.0 (Test item concentrations)

Dissolved oxygen:

8.8 - 9.0 mg/L (Control)
8.8 - 8.9 mg/L (Test item concentrations)

Nominal and measured concentrations:

Control, 6.25 12.5, 25, 50 and 100 mg test item/L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with glass plate to reduce the loss of water via evaporation)
- Material, size, headspace, fill volume: 100 mL glass beakers, 40 mL headspace
- Volume of solution: Approx. 60 mL of test medium
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt "M4" according to the OECD guideline 202; analytical grade salts and additives were added in deionised water
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 720 to 930 lux

EFFECT PARAMETERS MEASURED: Number of immobile organisms after 24 and 48 hours

RANGE-FINDING STUDY : Non-GLP range-finding test was performed

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

15.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidential limits

Remarks:

14.3 - 17.8 mg test item/L

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: No mortality was observed

Results with reference substance (positive control):

EC50 (24 h): 0.932 mg test item/L
EC50 (48 h): 0.672 mg test item/L
The results indicate that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

The 24-hour and 48-hour EC50 and the 95% confidence limits were calculated by the trimmed Spearman Karber procedure. The EC20 and EC10 cannot be determined with this method. The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data. The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Conclusion:

The toxic effect of the test item RCX 14-848 to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEL was determined to be 6.25 mg test item/L. The 48-hour LOEL was determined to be 12.5 mg test item/L and the 48-hour EL50 value was calculated to be 15.9 mg test item/L. The initial concentrations of the exposure concentrations during the test were verified in the analytical part. All reported results refer to loading rate because the test was dosed via the water accommodated fraction (WAF) approach.

Table 1: Influence of the test item on the mobility of Daphnia magna

Endpoint [mg test item/L]	24 hours	48 hours
EL50	> 100	15.9
95 % CI	n.d.	14.3 - 17.8
EL20	> 100	n.d.
95 % CI	n.d.	n.d.
EL10	> 100	n.d.
95 % CI	n.d.	n.d.
NOEL	12.5	6.25
LOEL	25.0	12.5

Values refer to nominal loading rates; CI: Confidence interval; n.d.: not determinable; NOEL and LOEL were determined directly from the raw data.

Table 2: Influence of RCX 14-848 on the Mobility of Daphnia magna and Sublethal Effects

Nominal Concentration mg test item/L]	No. of Daphnia tested	No. of immobilised Daphnia after		% immobilised Daphnia after		Observations
		24 h	48 h	24 h	48 h	24 h	48 h
Control	20	0	0	0	0	0	0
6.25	20	0	0	0	0	0	0
12.5	20	0	3	0	15	0	0
25	20	3	20	15	100	0	0
50	20	1	20	5	100	0	0
100	20	3	20	15	100	0	0

 

Table 3: Summary of Analytical Results

Sample Description [mg test item/L]	% of nominal	RSD [%]1)	n
Control	n.a.	n.d.	4
6.25	65	5	4
12.5	58	3	4
25	54	3	4
50	46	6	4
100	27	2	4

1) mean value of all measured samples per treatment group; RSD: relative standard deviation per treatment group; n: number of analysed samples; n.a.: not applicable

Table 4: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	0%	Yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	≥8.8 mg O2/L	Yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

EL50 (48 h): 15.9 mg/L (nominal, OECD 202, D. magna)

Additional information

There is one experimental study available, in which the short-term toxicity of the substance to aquatic invertebrates was investigated according to OECD guideline 202 and GLP.

In a static test, Daphnia magna were exposed to nominal test item loading rates of 6.25 12.5, 25, 50 and 100 mg test item/L for 48 h. Since the substance is a UVCB, the test solution was applied as water accommodated fraction (WAF). The actual test item concentrations were analytically verified via quantification of one component of the UVCB for fresh and aged samples by HPLC- UV detection. The initial measured concentrations ranged between 28 and 68% of nominal and the aged samples between 27 and 63% of nominal. After 48-hour NOEL was determined to be 6.25 mg test item/L based on the nominal loading rate. The 48-hour LOEL was determined to be 12.5 mg test item/L and the 48-hour EL50 value was calculated to be 15.9 mg test item/L, both endpoints were also based on the nominal loading rate.