3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium 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:

20 January 2017 to 03 March 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: At t = 0 and t = 48 h, 2.0 mL was sampled from the approximate centre of the test vessels. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Not applicable, analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch tested was completely soluble in test medium at the concentrations tested. Preparation of test solutions started with the highest concentration of 100 mg/L applying 16 to 27 minutes of magnetic stirring to accelerate dissolution of the test material in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The colour of the final test solutions ranged from light pink to red in the combined limit/range-finding test and from light pink to pink in the final test.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Straus, 1820, at least third generation
- Source: In-house laboratory culture with a known history; obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock (mean and range, SD): Daphnids originated from a healthy stock, 2nd to 5th brood. For the test, young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old were selected.
- Feeding during test: No

ACCLIMATION
- Acclimation period: Each batch is started with newborn daphnids, i.e. less than 3 days old, by placing about 250 into 5 litres of medium in an all-glass culture vessel. The maximum age of the cultures is 4 weeks. After 7 days of cultivation, half of the medium is renewed twice a week.
- Acclimation conditions (same as test or not): Yes. Daphnids were kept in medium M7 at 18 to 22 °C.
- Type and amount of food: A suspension of fresh water algae
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): Daphnia showed 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 °C

pH:

7.9 to 8.1

Dissolved oxygen:

8.9 to 9.1 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 0, 0.10, 0.22, 0.46, 1.0 and 2.2 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL test vessels
- Material, size, headspace, fill volume: All-glass vessels containing 80 mL of solution
- Aeration: No aeration of the test solutions
- 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: The following chemicals are dissolved in tap water purified by Reverse Osmosis (RO-water):
Adjusted 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, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
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 hours of light daily

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

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. No immobility was observed in the control and at the lowest test concentration during the test period. Effects were complete at the two highest test concentrations after 24 hours of exposure. No immobility was observed at 1.0 mg/L after 24 hours of exposure, while 80 % of the daphnids were immobile at the end of the test. The expected EC50 was between 0.10 and 1.0 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No daphnids became immobilised in the control and at the three lowest test concentrations throughout the test duration. A dose response was observed at the two highest test concentrations.
The responses recorded in this test allowed for reliable determination of an EC50. The 48 h-EC50 was 1.0 mg/L based on analytically confirmed nominal concentrations (95 % confidence interval between 0.85 and 1.2 mg/L).

ACCEPTABILITY OF THE TEST
1. 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.
2. The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels.

Results with reference substance (positive control):

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 within the range of 0.3 to 1.0 mg/L. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at the testing facility.
The 24 h EC50 was 0.64 mg/L with a 95 % confidence interval ranging from 0.56 to 0.73 mg/L.
The 48 h EC50 was 0.48 mg/L with a 95 % confidence interval ranging from 0.43 to 0.53 mg/L.

Reported statistics and error estimates:

Calculation of EC50: The 24 and 48 h-EC50-value was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding nominal test material concentrations (analytically confirmed) using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 1: Number of introduced daphnids and incidence of immobility in the final test

Time (h)	Replicate	Nominal concentration (mg/L)
		Control	0.10	0.22	0.46	1.0	2.2
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	1	5
	B	0	0	0	0	1	5
	C	0	0	0	0	1	5
	D	0	0	0	0	1	4
	Total immobilised	0	0	0	0	4	19
	Effect %	0	0	0	0	20	95
48	A	0	0	0	0	3	5
	B	0	0	0	0	3	5
	C	0	0	0	0	3	5
	D	0	0	0	0	2	5
	Total immobilised	0	0	0	0	11	20
	Effect %	0	0	0	0	55	100

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48 h-EC50 was 1.0 mg/L based on analytically confirmed nominal concentrations (95 % confidence interval between 0.85 and 1.2 mg/L).

Executive summary:

The potential for the test material to cause acute toxicity to Daphnia magna was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2 under GLP conditions.

The batch tested was completely soluble in test medium at the concentrations tested. A full test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate) were exposed to an untreated control and to nominal concentrations of 0.10, 0.22, 0.46, 1.0 and 2.2 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.

No daphnids became immobilised in the control and at the three lowest test concentrations throughout the test duration. A dose response was observed at the two highest test concentrations.

Analysis of the samples taken at the start and the end of the final test showed that measured concentrations were stable and in agreement with the nominal concentrations (92 to 105 %) throughout the test duration, with the exception of the concentrations measured in nominally 0.10 mg/L. The initially measured concentration in the sample from 0.10 mg/L was at the level of 29 % of nominal at the start of the exposure and at the level of 326 % of initial (i.e., 95 % of nominal) at the end of the test. Since such increase in test material concentration is unrealistic and all others samples showed that actual concentrations were at the level of nominal, the measurement at 24 hour in 0.10 mg/L was considered a mistake and effects parameters were expressed as analytically confirmed nominal concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the 48 h-EC50 was 1.0 mg/L based on analytically confirmed nominal concentrations (95 % confidence interval between 0.85 and 1.2 mg/L).

Description of key information

Under the conditions of this study, the 48 h-EC50 was 1.0 mg/L based on analytically confirmed nominal concentrations (95 % confidence interval between 0.85 and 1.2 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 mg/L

Additional information

The potential for the test material to cause acute toxicity to Daphnia magna 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).

The batch tested was completely soluble in test medium at the concentrations tested. A full test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate) were exposed to an untreated control and to nominal concentrations of 0.10, 0.22, 0.46, 1.0 and 2.2 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.

No daphnids became immobilised in the control and at the three lowest test concentrations throughout the test duration. A dose response was observed at the two highest test concentrations.

Analysis of the samples taken at the start and the end of the final test showed that measured concentrations were stable and in agreement with the nominal concentrations (92 to 105 %) throughout the test duration, with the exception of the concentrations measured in nominally 0.10 mg/L. The initially measured concentration in the sample from 0.10 mg/L was at the level of 29 % of nominal at the start of the exposure and at the level of 326 % of initial (i.e., 95 % of nominal) at the end of the test. Since such increase in test material concentration is unrealistic and all others samples showed that actual concentrations were at the level of nominal, the measurement at 24 hour in 0.10 mg/L was considered a mistake and effects parameters were expressed as analytically confirmed nominal concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the 48 h-EC50 was 1.0 mg/L based on analytically confirmed nominal concentrations (95 % confidence interval between 0.85 and 1.2 mg/L).