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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:

The experimental start date was 31 Jul 2017, and the experimental completion date was 16 Aug 2017.

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)

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assessment number 23, 2000

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h and t=48 h
Volume 40 mL from the approximate centre of the test vessels
Storage Samples were stored in a refrigerator (2 - 8°C) until analysis.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 40 mL were taken for possible analysis. If not used, these samples were stored in a refrigerator (2 - 8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: All test solutions were prepared separately at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. After a two-day period of magnetic stirring to reach maximum solubility of the test item in test medium, the resulting mixtures were left to stabilize for approximately 105-110 minutes, after which the Water Accommodated Fractions (WAFs) were siphoned off. All final test solutions were clear and colourless.

Any residual volumes were discarded unless otherwise requested by the Study Director.

- Controls: Test medium without test item or other additives.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All final test solutions were clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820
- Source: In-house laboratory culture with a known history.
- Age of parental stock (mean and range, SD): parental daphnids more than two weeks old.
- Feeding during test - none

ACCLIMATION
- Acclimation period: Not reported
- Acclimation conditions (same as test or not): Test medium different to culture medium
- Type and amount of food: Not reported
- Feeding frequency: Not reported
- Health during acclimation (any mortality observed): Breeding culture: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: For the test a selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Not reported

Test temperature:

20 - 21°C

pH:

8.6 - 9.4

Dissolved oxygen:

8.0 - 8.1

Salinity:

Freshwater

Conductivity:

Not reported

Nominal and measured concentrations:

WAF Loading Rate: 1.0, 3.2, 10, 32 and 100 mg/l

Initial Measured TOC: 0.015, 0.24, 1.6, 3.4, 8.5 mg/l

48 hour measured TOC: -0.0069, 0.066, 1.0, 2.8, 7.2 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass vessel
- Type (delete if not applicable): open / closed: not reported
- Material, size, headspace, fill volume: Glass, 100 ml filled with 80 ml of test solution.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Static
- Renewal rate of test solution (frequency/flow rate): Static
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium
- Culture medium different from test medium: Yes: culture medium was M7 Elendt medium
- Intervals of water quality measurement: Dissolved oxygen & pH: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.


OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16 hours photoperiod daily
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (including mortality): At 24 hours and at 48 hours.

VEHICLE CONTROL PERFORMED: yes/no: n/a

RANGE-FINDING STUDY: A final test was performed based on a preceding study (Test Facility project 510150).
- Test concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Results used to determine the conditions for the definitive study: 100% immobility observed at 1 mg/l. No immobility observed at higher concentrations, therefore test repeated including 1 mg/l concentration.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None reported
- Observations on body length and weight: not reported
- Other biological observations: none reported
- Mortality of control: No
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: n/a
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: 48h-EC50 was between 0.3 and 1.0 mg/l
- Limit test: No
- Dose-response test: Yes: Test concentrations 0.1, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l.
- ECx: 48h-EC50 0.59 mg/L with a 95% confidence interval between 0.52 and 0.66 mg/l.

Reported statistics and error estimates:

No EL50 could be calculated because the test item proved to be non-toxic (EL50 > maximum loading rate tested).

Table 1: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)	Replicate	Test substance (multi constituent); WAFLoading rate (mg/L)
		Control	1.0	3.2	10	32	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	0	0
	B	0	0	0	0	0	0
	C	0	0	0	0	0	0
	D	0	0	0	0	0	0 (1)
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0
48	A	0	0	0	0	0	0
	B	0	0	0	0	0	0
	C	0	0	0	0	0	0
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Validity criteria fulfilled:

yes

Conclusions:

A 48 hour EL50 value of >100 mg/l (nominal, WAF loading rate) (highest concentration tested) has been determined for the effects of the submission substance (CAS 504396-15-0) on mobility and mortality of Daphnia magna, in accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). In view of the test media preparation method/exposure regime it is likely that the test organisms were exposed predominantly to the hydrolysis products of each constituent of the tested substance.
It can furthermore be concluded that lower test item concentrations did not affect mobility of D. magna. The effect observed at a loading rate of 1.0 mg/L in a previous study with the test substance (multi constituent) (Test Facility Project 510150) was likely related to other causes and not exposure to the test item.

Description of key information

Short-term toxicity to freshwater invertebrates: EL₅₀ >100 mg/L (nominal, WAF loading rate) (highest concentration tested) for the effects of the submission substance on mobility of Daphnia magna in accordance with guideline OECD 202. The rapid hydrolysis rates of the constituents of the test substance means that the test organisms were predominantly exposed to the hydrolysis products of the test substance.

Additional information

A 48-hour EL₅₀ value of >100 mg/L (nominal, WAF loading rate) (highest concentration tested) has been determined for the effects of the submission substance (CAS 504396-15-0) on mobility and mortality of Daphnia magna, in accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). In view of the test media preparation method/exposure regime it is likely that the test organisms were exposed predominantly to the hydrolysis products of each constituent of the tested substance.

In an earlier experiment (Test Facility Project 510150), a NOEL of 100 mg/L was determined in a limit test for acute toxicity of the test item on D. magna. However, during the preceding combined limit/range-finding test of this study, a strong effect was found in a WAF prepared at a loading rate of 1.0 mg/L. Based on those findings, the test concentration range for this key study was selected to confirm whether or not the test item exhibits effects in WAFs prepared at loading rates below 100 mg/L.

No effects on mortality or mobility were observed in this test at any of the concentrations tested including the 1 mg/L concentration. It can therefore be concluded that lower test item concentrations did not affect mobility of D. magna. According to the study author, the effect observed at a loading rate of 1.0 mg/L in the previous, supporting, study with the submission substance (multi-constituent) (Test Facility Project 510150) was likely related to other causes and not exposure to the test item.

The supporting study (WIL Research, 2016) reported a 48-hour E(L)C₅₀ value of >100 mg/L (nominal, WAF) (highest concentration tested), determined for the effects of the submission substance (CAS 504396-15-0) on mobility and mortality of Daphnia magna. In view of the test media preparation method/exposure regime it is likely that the test organisms were exposed predominantly to the hydrolysis products of each constituent of the tested substance.

Although there were no effects in the highest concentration tested of 100 mg/L, during the range-finding test carried out prior to the definitive test, 100% immobility was observed in the lowest loading rate of 1 mg/L, but no effects were observed in the control or 10 and 100 mg/L loading rates. The study authors reported this as unexpected. The results of this test could not rule out that, at lower loading levels, this substance may be acutely toxic to Daphnia. The effect observed at 1 mg/L concentration could not be dismissed, therefore the study was repeated to see if the effects were due to test substance or another, unexplained cause. A second test was therefore conducted (discussed above), that included concentrations at 1 mg/L WAF loading rate. No effects were observed.