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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 Okt 2020 - 22 Apr 2021

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:

Apr 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Range finding:
- Concentrations: 0, 1.0, 10, 100 mg/L (nominal water accommodated fraction (WAF) loading rate)
- Sampling method: Water samples were taken from each WAF loading rate at 0 and 48 h for quantitative analysis. A duplicate sample was frozen for further analysis if required.
- Sample storage conditions before analysis: Analysis occurred immediately, unless the duplicate sample was analysed. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Initial test:
- No analysis of the test item

Definitive test:
- Concentrations: 0, 1.0, 3.2, 10, 32, 100 mg/L (nominal WAF loading rate)
- Sampling method: Water samples were taken from each freshly prepared loading rate at 0 and 24 h and from the corresponding aged preparation at 24 and 48 h for quantitative analysis (replicates pooled). A duplicate sample was frozen for further analysis if required.
- Sample storage conditions before analysis: Analysis occurred immediately, unless the duplicate sample was analysed. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Since the test substance is a complex mixture (UVCB) and is poorly soluble in water, organisms were exposed to water accommodated fractions (WAF) of the test item.
- Method: WAF

VALIDATION OF MIXING PERIOD:
- Loading rate: 100 mg/L (n=2)
- Stirring rate: stirring rate such that a vortex was formed to give a dimple at the water surface
- Duration of stirring: treatment 1: 23 h (n=1); treatment 2: 95 h (n=1)
- Standing periode: 1 h
- Sampling time: treatment 1: 24 h; treatment 2: 96 h
- Sampling method: The aqueous phase was removed by filtration through glass wool and filter paper.
- Test medium: Elendt M4

Results showed that increasing the stirring period did not increase the amount of the test item in the WAF and so the stirring period of the WAF was maintained at 24 h.

PREPERATION OF WAF FOR THE RANGE FINDING TEST
- Loading rate WAF: 1.0, 10 and 100 mg/L (n=2)
- Description: Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2.0 and 2.0 L of test water.
- Stirring rate: stirring rate such that a vortex was formed to give a dimple at the water surface
- Duration of stirring: 23 h
- Standing periode: 1 h
- Test medium: Elendt M4
- Sampling time: 24 h
- Sampling method: The aqueous phase was removed by mid-depth siphoning (the first 75 to 100 ml were discarded) and filtration through glass wool and filter paper. The 100 mg/L loading rate was filtered twice through a filter paper.
- Test concentration separation factor: 10
- Evidence of undissolved material: Microscopic observations of the WAFs showed no presence of micro-dispersions of the test item in the 1.0 and 10 mg/L loading rate. However, test item was still observed in the 100 mg/L loading rate.

PREPERATION OF WAF FOR THE INITIAL EXPERIMENT
- Loading rate WAF: 1.0, 1.8, 3.2, 5.6 and 10 mg/L
- Stirring rate: stirring rate such that a vortex was formed to give a dimple at the water surface
- Duration of stirring: 23 h
- Standing period: 1 h
- Test medium: Elendt M4
- Sampling time: 24 h
- Sampling method: The aqueous phase was removed by mid-depth siphoning (the first 75 to 100 ml were discarded) and filtration through glass wool and filter paper.
- Test concentration separation factor: approx. 1.8


PREPERATION OF WAF FOR THE DEFINITIVE TEST
- Loading rate WAF: 1.0, 3.2, 10, 32 and 100 mg/L (n=4)
- Controls WAF: 0 mg/L
- Description: Nominal amounts of the test item were each separately added to the surface of 10, 5.0, 5.0, 5.0 and 5.0 L of test water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. A control WAF was also prepared using 2.0 L of test water.
- Stirring rate: stirring rate such that a vortex was formed to give a dimple at the water surface
- Duration of stirring: 23 h
- Standing period: 1 h
- Test medium: Elendt M4
- Sampling time: 24 h
- Sampling method: The aqueous phase was removed by mid-depth siphoning (the first 75 to 100 ml were discarded) and filtration through glass wool and filter paper
- Test concentration separation factor: approx. 3.2
- Evidence of undissolved material: Microscopic observations of the WAFs showed no presence of micro-dispersions of the test item in all loading rates of the definitive test.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: first instar, age < 24 h
- Source: in-house laboratory culture
- Feeding during test: no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
- Method of breeding: Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
- Temperature: temperature controlled room, 18-22 °C
- Light cycle: 16 h light and 8 h darkness, with 20 min dawn and dusk transition periods
- Feeding: Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

19 - 21 °C

pH:

7.7 - 8.3

Dissolved oxygen:

8.5 - 9.1 mg/L

Nominal and measured concentrations:

nominal, WAF loading rate: 0, 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 ml glass vessels
- Type: covered
- Volume of solution: 100 mL
- Aeration: no
- Renewal rate of test solution: after 24 h
- 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
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water temperature, pH and dissolved oxygen were recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: not during the test
- Photoperiod: 16 h light, 8 h dark with 20 min dusk to dawn transition period
- Light intensity: 200 - 1200 lux

EFFECT PARAMETERS MEASURED:
- Mobility: every 24 h
- Any other adverse reactions to exposure: every 24 h

WAF CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L (nominal, WAF loading rates)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate; Tests were performed twice in a 12 month period.; 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L (nominal, potassium dichromate). The positive control was conducted between 05 January 2021 and 29 January 2021.

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

4.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

mobility

Remarks on result:

not determinable because of methodological limitations

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

mobility

Remarks on result:

not determinable because of methodological limitations

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

mobility

Remarks on result:

not determinable because of methodological limitations

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

mobility

Remarks on result:

not determinable because of methodological limitations

Duration:

24 h

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

mobility

Remarks on result:

not determinable because of methodological limitations

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

mobility

Remarks on result:

not determinable because of methodological limitations

Details on results:

A sub-lethal effect was observed in 3.2 mg/L WAF loading rate: 15% reduced mobility after 24 h

Results with reference substance (positive control):

24 h EC50: 0.86 mg/L (nominal)
48 h EC50: 0.50 mg/L (nominal)

Reported statistics and error estimates:

The EL50 values and associated confidence limits at 24 and 48 hours and the slope of the
response curve and its standard error were calculated by Weibull analysis using Linear
Maximum-Likelihood regression. The Lowest Observed Effect Loading (LOEL) rate and the
No Observed Effect Loading (NOEL) rate at 24 and 48 hours were calculated using the
Multiple Sequentially-rejective Chi2-2x2-Table test After Bonferroni-Holm incorporating
Tarone’s test and Qualitative trend Analysis by Contrasts. All results were calculated using
the ToxRat computer software package (ToxRat® Solutions GMBH).

VALIDATION OF MIXING PERIOD

Increasing of the stirring period from 24 h to 96 h did not increase the amount of the test item in the WAF (54.8 mg/L and 50.5 mg/L fatty acids at a nominal WAF loading rate of 100 mg/L) and so preparation of the WAF was maintained at 24 h.

 

PRELIMINARY TEST

- Significant immobilization observed at 10 and 100 mg/L WAF loading rate

- Measured test concentrations ranged from 0.23 to 49 mg/L at 0 h and 0.11 to 39 mg/L at 48 h indicating that the test item was not stable under test conditions.

 

INITIAL TEST

- 15% immobilization at 10 mg/L WAF loading rate after 48 h

- EL50: n.c.

 

RESULTS OF THE DEFINITIVE TEST

Table 1           Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Loading Rate (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5 N	5 N	5 N
1.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	1	0	2	0	3	15	4 N	3 N 2 R	3 N	4 N 1 R
10	5	5	5	5	20	100	AI	AI	AI	AI
32	5	5	5	5	20	100	AI	AI	AI	AI
100	5	5	5	5	20	100	AI	AI	AI	AI

Nominal Loading Rate (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	1	2	3	0	6	30	4 N	3 N	2 N	5 N
10	5	5	5	5	20	100	AI	AI	AI	AI
32	5	5	5	5	20	100	AI	AI	AI	AI
100	5	5	5	5	20	100	AI	AI	AI	AI

Rx      = Replicate

N        = Normal

R        = Reduced mobility

AI       = All Daphnia immobilized

VALIDITY CRITERIA DEFINITIVE TEST:

OECD 202: Short-term toxicity to aquatic invertebrates

Table 1: Validity criteria for OECD 202 (2004)

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% immobility	yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	8.5 - 9.1 mg/L	yes

VERIFICATION OF MEASURED CONCENTRATIONS - DEFINITIVE TEST (see also Table 2 below)

- 0 and 24 h (1.0 - 100 mg/L, nominal WAF loading rate): 0.28 - 39 mg/L (measured)

- 24 and 48 h (1.0 - 100 mg/L, nominal WAF loading rate): 0.17 - 38 mg/L (measured)

Table 2 Analytical results – Definitive test (Analyte: Fatty acids)

Sample description	Time point (h)	Analyte detected (mg/L)	Nominal recovery (% LR)
Control	0	< LOD	-
1.0 mg/L LR WAF		0.294	29
3.2 mg/L LR WAF		0.677	21
10 mg/L LR WAF		6.71	67
32 mg/L LR WAF		21.3	67
100 mg/L LR WAF		38.6	39
Control	24 (old)	< LOD	-
1.0 mg/L LR WAF		0.200	20
3.2 mg/L LR WAF		0.455	14
10 mg/L LR WAF		2.77	28
32 mg/L LR WAF		15.9	50
100 mg/L LR WAF		38.2	38
Control	24 (new)	< LOD	-
1.0 mg/L LR WAF		0.279	28
3.2 mg/L LR WAF		2.16	68
10 mg/L LR WAF		8.62	86
32 mg/L LR WAF		25.2	79
100 mg/L LR WAF		30.9	31
Control	48	< LOD	-
1.0 mg/L LR WAF		0.165	17
3.2 mg/L LR WAF		0.810	25
10 mg/L LR WAF		5.89	59
32 mg/L LR WAF		23.0	72
100 mg/L LR WAF		30.6	31

LR:        loading rate

WAF:   water accommodated fraction

< LOD: less than the limit of detection of 3 ng/mL (equivalent to 0.006 mg/L in initial sample matrix)

-:          not applicable

 

Procedural recovery results for definitive test (1 and 10 mg/L, nominal, n=3): 100-107%

The dissolved test item may have been one or several components of the test item. Given that
the toxicity cannot be attributed to a single component or a mixture of components, but to the
test item as a whole, the results were based on nominal loading rates only.

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

Short-term toxicity to aquatic invertebrates:

EL50 (48 h) = 4.1 mg/L for Daphnia magna (nominal, WAF loading rate, OECD 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

The EC50 is an EL50

Effect concentration:

4.1 mg/L

Additional information

The available key study on short-term toxicity of the UVCB test substance to aquatic invertebrates was performed according to OECD guideline 202 (GLP) with neonates (age < 24 h) of Daphnia magna. Organisms were exposed to individually prepared water accommodated fractions (WAF) of the UVCB test substance of 0, 1.0, 3.2, 10, 32 and 100 mg/L (nominal, WAF loading rate) under semi-static conditions for 48 h. Analytical determination of test solution concentration was performed by analyzing fatty acid concentrations in the WAFs as an surrogate for the UVCB test substance. Chemical analysis resulted in low concentrations of fatty acids of 0.28 - 39 mg/L in freshly prepared medium and 0.17 - 38 mg/L before test medium renewal periods after 24 or 48 h, respectively. Since the toxicity cannot be linked to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only. Concentration dependent effects on mobility were observed after 48 h, resulting in an EL50 of 4.1 mg/L (nominal, WAF loading rate).