Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Testing was conducted between the 4th March 2018 and the 21st March 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: Pigment Red 81:4
CAS Number: 85959-61-1
Batch: 64078
Purity: 97 to 100%
Physical state/Appearance: Red powder
Expiry Date: 30 June 2018
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal concentration of 10 mg/L
- Sampling method: The test samples were thawed in a water bath. The test item was extracted from the test samples using a solid phase extraction cartridge (Strata C18-E, 500 mg/3 mL). The cartridge was pre-conditioned with 10 mL of acetonitrile and 10 mL of 1% (v/v) phosphoric acid in water. The samples (100 mL) were drawn through the cartridge under reduced pressure. Subsequently, the cartridge was eluted with 10 mL of 1% (v/v) phosphoric acid in acetonitrile, into a 20 mL volumetric flask. The solution was made up to the mark with water.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis

Vehicle:

yes

Details on test solutions:

The test water used for the limit test was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given below:

Analysis Typical Value Units
Hardness Level Slightly Hard na
Hardness clark 8.76 Degrees clark
Hardness french 12.52 French degrees
Hardness german 7.01 German degrees
Aluminium 7.46 mg Al/L
Chlorine 0.32 mg/L
Coliform bacteria 0 no./100mL
Colour 0.6 mg/l Pt/Co
Conductivity 345 μS/cm at 20 °C
E.Coli bacteria 0 no./100mL
Fluoride 0.24 mg F/L
Iron 19.9 μg Fe/L
Manganese 2.9 μg Mn/L
Nitrate 7.61 mg NO3/L
Odour 0 Dilution number
Pesticides 0 μg/L
pH 7.66 pH Value
Sodium 22.35 mg Na/L
Taste 0 Dilution number

Plumbing Metals Typical Value Units
Copper 0.02 mg Cu/L
Lead 0.51 μg Pb/L
Nickel 3.1 μg Ni/L

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 18 December 2017. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. F

ACCLIMATION :
ish were acclimatized to test conditions from 26 February 2018 to 05 March 2018. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.7 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the limit test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.9 cm (standard deviation = 0.43) and a mean body weight of 1.7 g (standard deviation = 0.39) at the end of the limit test. Based on the mean body weight value this gave a loading rate of 0.60 g body weight/liter.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

The water had a total hardness of approximately 140 mg/L as CaCO3.

Test temperature:

The test vessels were then covered to reduce evaporation and maintained at 13 °C to 15 °C in a temperature controlled room. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The pH was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24 hour old test preparations. The pH was measured using a Hach Flexi handheld meter.

Dissolved oxygen:

The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24 hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Salinity:

Not applicable as a fresh water study was undertaken.

Conductivity:

Not applicable

Nominal and measured concentrations:

A nominal concentration of 10 mg/L was used within the limit test.
Due to the low water solubility of the test item, the mean measured test concentrations ranged from between 0.32 to 0.35 mg/L.

Details on test conditions:

TEST ITEM PREPARATION:
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 0.68 mg/L was obtained using a preliminary solution in dechlorinated tap water.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

EXPERIMENTAL PREPARATION:
A nominal amount of test item (2200 mg) was dispersed in 22 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre condition the filter) to give a 100% v/v saturated solution.
The stock solution was mixed with a flat bladed stirrer for approximately 1 minute to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours and of the old media at 24 and 96 hours

TEST SYSTEM :
- For each control and test concentration, 25 to 30 liter glass exposure vessels containing 20 liters of test media were used. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 13 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.
A semi static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build up of nitrogenous waste products.

OTHER TEST CONDITIONS :
with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any mortalities and sub lethal effects of exposure were recorded at 1, 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS :
A nominal concentration of 10 mg/L was used within the limit test.
Due to the low water solubility of the test item, the mean measured test concentrations ranged from between 0.32 to 0.35 mg/L.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Limit Test:
Verification of Test Concentrations:
Analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations to range from 0.32 to 0.35 mg/L. Analysis of the old or expired test preparations at 24 hours showed a measured test concentration of 0.35 mg/L. A decline in measured test concentration was observed at 96 hours to 0.24 mg/L and hence it was considered appropriate to calculate the results based on the mean measured test concentration only in order to give a “worst case” analysis of the data.

The geometric mean measured test concentrations were determined to be:
Nominal Test Concentration (% v/v Saturated Solution) Geometric Mean Measured Test Concentration (mg/L)
100 0.31

Sublethal observations / clinical signs:

Mortality Data:

Cumulative mortality data from the exposure of rainbow trout to the test item during the limit test are given in the table below:

Nominal Concentration (% v/v Saturated Solution)	Cumulative Mortality (Initial Population = seven) (Hours)							Mortality (%)
	1	3	6	24	48	72	96	96 Hours
Control	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0

There were no mortalities of the seven fish exposed to a geometric mean test concentration of 0.31 mg/L for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (hour)	LC50 (mg/L)
1	>0.31
3	>0.31
6	>0.31
24	>0.31
48	>0.31
72	>0.31
96	>0.31

 

The results of the limit test showed the highest test concentration resulting in 0% mortality to be 0.31 mg/L. The NOEC was 0.31 mg/L. 

Sub-Lethal Effects:

Sub‑lethal effects of exposure are provided in the table below:

Sub-lethal Effects of Exposure

Nominal Concentration (% v/v Saturated Solution)	Sub-lethal Effects	Time (Hours)
		1	3	6	24	48	72	96
Control	No abnormalities detected	0	0	0	0	0	0	0
100	No abnormalities detected	0	0	0	0	0	0	0

There were no sub‑lethal effects of exposure observed in the test.

Validation Criteria:

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O₂/L) in the control and test vessels.

Water Quality Criteria:

The results of the water quality measurements are provided in the table below:

Water Quality Measurements:

Nominal Concentration (% v/v Saturated Solution)		Time (Hours)
		0 Hours (Fresh Media)				24 Hours (Old Media)
		pH		mg O2/L		Temperature °C		pH		mg O2/L		Temperature °C
Control		8.0		11.7		14		8.2		9.3		13
100		7.7		10.3		15		7.8		9.2		14
Nominal Concentration (% v/v Saturated Solution)	Time (Hours)
	24 Hours (Fresh Media)			48 Hours (Old Media)
	pH	mg O2/L	Temperature °C	pH	Temperature °C	Temperature °C
Control	7.4	11.8	14	7.9	9.4	13
100	7.8	10.1	15	8.2	9.7	14
Nominal Concentration (% v/v Saturated Solution)	Time (Hours)
	48 Hours (Fresh Media)			72 Hours (Old Media)
	pH	mg O2/L	Temperature °C	pH	mg O2/L	Temperature °C
Control	7.7	11.3	14	7.4	9.6	13
100	7.7	10.1	14	7.6	9.8	14
Nominal Concentration (% v/v Saturated Solution)	Time (Hours)
	72 Hours (Fresh Media)			96 Hours (Old Media)
	pH	mg O2/L	Temperature °C	pH	mg O2/L	Temperature °C
Control	7.6	11.1	14	7.3	9.9	13
100	7.4	10.2	14	7.5	9.7	14

 

Temperature was maintained at 13 °C to 15 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Observations on Test Item Solubility:

The test item preparations were observed to be clear orange/pink solutions throughout the test.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the geometric mean measured test concentrations gave a 96 hour LC50 of greater than 0.31 mg/L. The NOEC was 0.31 mg/L. This study showed that there were no toxic effects at saturation.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods:

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.68 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of 13 ºC to 15 °C under semi‑static test conditions. The test item solution was prepared by stirring an excess (10 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µmSartorius Sartopore filter, first approximate 2 liters discarded in order to pre‑condition the filter) to produce a saturated solution of the test item with a nominal concentration of 10 mg/L. The number of mortalities and any sub‑lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results:….

Analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations to range from 0.32 to 0.35 mg/L. Analysis of the old or expired test preparations at 24 hours showed a measured test concentration of 0.35 mg/L. A decline in measured test concentration was observed at 96 hours to 0.24 mg/L and hence it was considered appropriate to calculate the results based on the mean measured test concentration only in order to give a “worst case” analysis of the data.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC₅₀ values based on the geometric mean measured test concentrations of greater than 0.31 mg/L. The No Observed Effect Concentration (NOEC) was 0.31 mg/L.

This study showed that there were no toxic effects at saturation.

Description of key information

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC₅₀values based on the geometric mean measured test concentrations of greater than 0.31 mg/L. The No Observed Effect Concentration (NOEC) was 0.31 mg/L. This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.31 mg/L

Additional information

Introduction:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods:

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.68 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of 13 ºC to 15 °C under semi‑static test conditions. The test item solution was prepared by stirring an excess (10 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µmSartorius Sartopore filter, first approximate 2 liters discarded in order to pre‑condition the filter) to produce a saturated solution of the test item with a nominal concentration of 10 mg/L. The number of mortalities and any sub‑lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results:….

Analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations to range from 0.32 to 0.35 mg/L. Analysis of the old or expired test preparations at 24 hours showed a measured test concentration of 0.35 mg/L. A decline in measured test concentration was observed at 96 hours to 0.24 mg/L and hence it was considered appropriate to calculate the results based on the mean measured test concentration only in order to give a “worst case” analysis of the data.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC₅₀values based on the geometric mean measured test concentrations of greater than 0.31 mg/L. The No Observed Effect Concentration (NOEC) was 0.31 mg/L.

This study showed that there were no toxic effects at saturation.