3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]xanthylium chloride

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:

10 March 2017 to 30 August 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)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for possible analysis were taken from all test concentrations and the control.
- Sampling method: 1.2 mL from the approximate centre of the test vessels was taken at t = 0 and t = 48 h.
- Sample storage conditions before analysis: Not applicable, samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test material was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition. Preparation of test solutions started with a concentration of 100 mg/L applying an overnight period of magnetic stirring to accelerate dissolution of the test material in medium. Lower test concentrations were prepared by subsequent dilutions of the concentration of 100 mg/L in test medium. All test solutions were clear and ranged from light pink to red at the end of the preparation procedure.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Crustacea, Cladocera, Straus 1820
- Source: In-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age: For the test young daphnids with an age of < 24 hours were selected, from parental daphnids of more than two weeks old.
- Feeding during test: No

ACCLIMATION
- Acclimation conditions: Each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. The maximum age of the cultures was 4 weeks; after 7 days of cultivation half of the medium was renewed twice a week. Temperature of the medium was 18 - 22 °C. Medium was M7.
- Type and amount of food: A suspension of fresh water algae
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

20 to 21 ° C

pH:

7.8 to 8.0

Dissolved oxygen:

8.9 to 9.0 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 0.010, 0.022, 0.046, 0.10 and 0.22 mg/L
- Average measured exposure concentrations: 0.0076, 0.017, 0.036, 0.086 and 0.18 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL all glass vessel
- Material, size, headspace, fill volume: 50 mL per vessel
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 5 organisms per 50 mL of solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7 medium was prepared by addition of trace elements, macronutirnets and vitamins to freshly prepared ISO medium. The required chemicals were dissolved in tap water purified by Reverse Osmosis (RO-water).
ISO medium: Macro salts: CaCl2.2H2O 211.5 mg/L, MgSO4.7H2O 88.8 mg/L, NaHCO3 46.7 mg/L and KCl 4.2 mg/L.
Medium M7: Trace elements: B 0.125 mg/L, Fe 0.05 mg/L, Mn 0.025 mg/L, Li, Rb and Sr 0.0125 mg/L, Mo 0.0063 mg/L, Br 0.0025 mg/L, Cu 0.0016 mg/L, Zn 0.0063 mg/L, Co and I 0.0025 mg/L, Se 0.0010 mg/L, V 0.0003 mg/L and Na2EDTA.2H2O 2.5 mg/L. Macro nutrients: Na2SiO3.9H2O 10.0 mg/L, NaNO3 0.27 mg/L, KH2PO4 0.14 mg/L and K2HPO4 0.18 mg/L. Vitamins: Thiamine 75.0 μg/L, B12 1.0 μg/L and Biotin 0.75 μg/L.
- Culture medium different from test medium: Daphnids were cultured in medium M7 and the test carried out in adjusted ISO medium.
- Intervals of water quality measurement: At the beginning and at the end of the test, pH and dissolved oxygen were determined. Temperature of medium was monitored continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hour photoperiod daily

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

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
The study started with a combined limit/range-finding test. As the toxicity was below the range tested, a range-finding test was performed with a lower concentration range. Thereafter two full tests were performed.
- Test concentrations: Combined limit/range-finding test: 0, 0.1, 1.0, 10 and 100 mg/L. Range-finding test: 0, 0.0010, 0.010, 0.10 and 1.0 mg/L.
- Results used to determine the conditions for the definitive study: Yes. In the combined limit/ range-finding test no immobility was observed in the control during the exposure period, while all daphnids were immobilised in all test material treated groups at the end of the exposure. In the range-finding test, no immobility was observed in the control and the two lowest concentrations tested during the exposure period. All daphnids were immobilised in the highest concentration after the first 24 hours of exposure, while 50 % of daphnids were immobilised in the 0.10 mg/L at the end of the test. Therefore, the expected EC50 approximated 0.10 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CI: 0.11 to 0.13 mg/L

Details on results:

FIRST FULL TEST
- Measured concentrations: The measured concentrations increased with the nominal concentration at the start of the test from 0.011 mg/L in the lowest group to 1.2 mg/L in the highest group tested (i.e. were between 108 and 120 % of nominal). The concentrations were at the level of 47 to 98 % of nominal at the end of the test. It should be noted that small responses were detected in the control treatment. They were considered to result from carry-over and therefore have no impact on the results.
- Immobility: No immobility was observed in the control during the exposure period. A dose-related response was observed at the average exposure concentrations of 0.018 to 1.0 mg/L, however, a not dose-related effect was observed at the lowest concentration tested. As the measured concentrations increased with the nominal, they did not explain the observed immobilisation pattern. For this reason, the results of this test were considered to be not reliable and the full test was repeated.

FINAL TEST
- Measured concentrations: The actual concentrations increased with the nominal concentrations at the start of the test. At the end of the exposure the actual concentrations were at the level of 49 to 107 % of initially measured. Therefore, the average exposure concentrations were calculated (see Table 1). It should be noted that small responses were detected in the control treatment. They were considered to result from carry-over and therefore, have not impact on the results.
- Immobility: Table 2 shows the responses recorded during the final test. The responses recorded in this test allowed for reliable determination of an EC50. No significant immobility (≥ 10 %) was observed in any of the groups tested but the highest concentration at the end of the exposure period.

ACCEPTABILITY OF THE TEST
- In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
- The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
The reference test was carried out to check the sensitivity of the test system as used by the testing facility. Daphnids were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration.
The actual responses in this reference test were within the ranges of the expected responses at the different concentrations, i.e. the 48 h EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of Daphnia magna was in agreement with the historical data collected at the testing facility.
The 24 h EC50 was 0.95 mg/L with a 95 % confidence interval ranging from 0.85 to 1.1 mg/L. The 48 h EC50 was 0.59 mg/L with a 95 % confidence interval ranging from 0.52 to 0.66 mg/L.

Reported statistics and error estimates:

Calculation of EC50: The 24 h EC50 could not be determined because no effects were observed at this time point. The 48 h EC50-value was calculated with the Spearman-Karber method. ToxRat Professional v 3.2.1. was used to perform the analyses.

Table 1: Average exposure concentration versus nominal concentration during the final test

Nominal concentration (mg/L)	Measured concentrations (mg/L)		Average exposure concentrations (mg/L)
	t = 0 h	t = 48 h
0.010	0.011	0.0054	0.0076
0.022	0.023	0.013	0.017
0.046	0.048	0.027	0.036
0.10	0.099	0.075	0.086
0.22	0.18	0.19	0.18

Table 2: Number of introduced daphnids and incidence of immobility in the final test

Time (h)	Replicate	Average exposure concentration of test material (mg/L)
		Control	0.0076	0.017	0.036	0.086	0.18
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
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0
48	A	0	0	0	0	1	5
	B	0	0	0	0	0	5
	C	0	0	0	0	0	5
	D	0	0	0	0	0	5
	Total immobilised	0	0	0	0	1	20
	Effect %	0	0	0	0	5	100

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48 h EC50 was 0.12 mg/L based on average exposure concentrations (95 % confidence interval between 0.11 and 0.13 mg/L).

Executive summary:

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions.

An acute immobilisation test was performed on Daphnia magna under static conditions. The test material was completely soluble in test medium at the concentrations tested. A full test was performed based on the results of preceding range-finding tests. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to nominal concentrations of 0.010, 0.022, 0.046, 0.10 and 0.22 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all groups were analysed. The actual concentrations increased with the nominal concentrations at the start of the test. At the end of the exposure the actual concentrations were at the level of 49-107 % of initially measured. Based on these results the average exposure concentrations were 0.0076, 0.017, 0.036, 0.086 and 0.18 mg/L for the nominal concentrations of 0.010, 0.022, 0.046, 0.10 and 0.22 mg/L, respectively.

No significant immobility (≥ 10 %) was observed in any of the groups tested but the highest concentration at the end of the exposure period. The study met the acceptability criteria prescribed by the study plan and was considered valid. The responses recorded in the test allowed for reliable determination of an EC50.

Under the conditions of this study, the 48 h EC50 was 0.12 mg/L based on average exposure concentrations (95 % confidence interval between 0.11 and 0.13 mg/L).

Description of key information

Under the conditions of this study, the 48 h EC50 was 0.12 mg/L based on average exposure concentrations (95 % confidence interval between 0.11 and 0.13 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.12 mg/L

Additional information

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

An acute immobilisation test was performed on Daphnia magna under static conditions. The test material was completely soluble in test medium at the concentrations tested. A full test was performed based on the results of preceding range-finding tests. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to nominal concentrations of 0.010, 0.022, 0.046, 0.10 and 0.22 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all groups were analysed. The actual concentrations increased with the nominal concentrations at the start of the test. At the end of the exposure the actual concentrations were at the level of 49-107 % of initially measured. Based on these results the average exposure concentrations were 0.0076, 0.017, 0.036, 0.086 and 0.18 mg/L for the nominal concentrations of 0.010, 0.022, 0.046, 0.10 and 0.22 mg/L, respectively.

No significant immobility (≥ 10 %) was observed in any of the groups tested but the highest concentration at the end of the exposure period. The study met the acceptability criteria prescribed by the study plan and was considered valid. The responses recorded in the test allowed for reliable determination of an EC50.

Under the conditions of this study, the 48 h EC50 was 0.12 mg/L based on average exposure concentrations (95 % confidence interval between 0.11 and 0.13 mg/L).