[No public or meaningful name is available]

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 07, 2018 to February 16, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 236 (Fish embryo acute toxicity (FET) test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

To demonstrate that nominal exposure concentrations were being achieved the concentrations of test substance in the test vessels were measured using the high-performance liquid chromatography method.

Details on sampling:

At study start, samples were taken from excess test solutions and at study end from pooled test vessel replicates of the dilution water control and each test concentration.

Vehicle:

yes

Remarks:

Water

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test organisms were newly fertilised embryos of unexposed wild-type Tübingen zebrafish, Danio rerio, obtained from continuous laboratory cultures held at Scymaris. The broodstock of zebrafish were free from infection and disease and had not undergone any pharmaceutical (acute or prophylactic) treatment for >2 months before spawning. The zebrafish brood stock was maintained in dechlorinated water, the same as the test dilution water, at a temperature of 26 ± 1°C. A photoperiod of 16 h light:8 h dark, with 20 minute transition periods was provided. Pairs were set up the night before the study in spawning chambers with dividers. Dividers were removed in the morning and eggs collected. To avoid genetic bias, eggs were collected from a minimum of three breeding pairs, mixed and impartially selected. The eggs were washed with dilution water after collection from the spawning chambers. The fertilisation rate of the eggs collected from 5 pairs was approximately 80 to 95%. The embryos were immersed in the test solutions within 90 minutes after the dividers were removed to ensure the embryos were exposed, at latest, by the 16 cell-stage.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

26 ± 1ºC

pH:

7.97 to 8.11

Dissolved oxygen:

87.7 to 97.2% of saturation

Nominal and measured concentrations:

Control and 1.25, 2.5, 5 10 and 20 mg/L (nominal)
Control and 0.24, 1.0, 2.7, 7.7 and 16 mg/L (mean measured)
The test concentration range was based on the lack of toxicity and dispersibility of the test substance demonstrated in a non-GLP range-finding test.

Details on test conditions:

The test vessels were 24-well plates, containing a nominal 2 mL of solution per well. Twenty replicates per test concentration were employed with four internal plate controls, the control consisted of twenty-four replicates. The well plates were covered with loose fitting lids. The positions of the treatments were randomly allocated within the test area.

The study was run with a dilution water control, solvent control and positive control together with nominal exposure concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L. Levels were based on the level of achievable solubility.

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline (Supplier: Acros organics, Lot/batch number: A0336829, Purity: 99.6%, Certificate of Analysis re-test date: January 2021, Sample storage: Room temperature

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Calculation method: Direct observation from data

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Calculation method: Direct observation from data

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Calculation method: Direct observation from data

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Calculation method: Direct observation from data

Results with reference substance (positive control):

The positive control had 100% mortality at 96 h.

Sublethal observations / clinical signs:

Results

Analytical data

The limit of quantification (LOQ) of test substance in thisstudy was 0.2 mg/L for all concentrations with the exception of the nominal 20 mg/L which was 0.4 mg/L. The instrument LOQ was 0.1 mg/L but during analysis, samples from the control and each test concentration were diluted ×2, doubling the LOQ, with the exception of the nominal 20 mg/L which was diluted x4. Analytical values are quoted to two significant figures and percentages to the nearest integer. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R² value of 0.99. The maximum percentage difference from nominal concentration for standards at the LOQ was less than 30% and less than 20% at levels greater than the LOQ. The measured concentration at the start of the study ranged from 110 - 124% of nominal. The measured concentration at the end of the study ranged from 3 - 60% of nominal.On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

The following are the results of the analysis in Table 1:

Nominal concentration of test substance (mg/L)	Measured concentration of test substance (mg/L)				Mean measured concentration (mg/L)	Mean measured concentration (%)
	0 h		96 h
	(mg/L)	% of nominal	(mg/L)	% of nominal
Control	<LOQ	-	<LOQ	-	0	-
1.25	1.5^a	117	0.040^b	3	0.24	19
2.5	3.1	124	0.33	13	1.0	40
5	5.8	116	1.3	26	2.7	55
10	12	120	4.9	49	7.7	77
20	22	110	12	60	16	81

All measurements are quoted to 2 significant figures. Geometric means are used to calculate mean measured concentrations.

^a Triplicate analyses: 1.5, 1.4, 1.5 mg/L

^b Triplicate analyses: 0.051, 0.033, 0.036 mg/L

The limit of quantification (LOQ) in thestudy was 0.2 mg/L for all concentrations with the exception of the nominal 20 mg/L which was 0.4 mg/L. The instrument LOQ was 0.1 mg/L but during analysis, samples were diluted ×2, doubling the LOQ, with the exception of the nominal 20 mg/L which was diluted x4. Values in Table 1 have been corrected for these dilutions.

Biological data

The numbers of zebrafish mortalities after 24, 48 72 and 96 h are given in Table 2:

Table 2: Embryo mortality and hatching observations

Time (h)	Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Number of mortalities per treatment	Total number tested	Percentage mortality	Percentage hatched*
24	Control	0	0	24	0	0
	1.25	0.24	2	20	10	0
	2.5	1.0	0	20	0	0
	5	2.7	0	20	0	0
	10	7.7	0	20	0	0
	20	16	0	20	0	0
48	Control	0	0	24	0	0
	1.25	0.24	2	20	10	17
	2.5	1.0	2	20	10	0
	5	2.7	0	20	0	0
	10	7.7	0	20	0	0
	20	16	0	20	0	25
72	Control	0	1	24	4	91
	1.25	0.24	2	20	10	94
	2.5	1.0	2	20	10	89
	5	2.7	0	20	0	90
	10	7.7	0	20	0	75
	20	16	0	20	0	85
96	Control	0	1	24	4	100
	1.25	0.24	2	20	10	100
	2.5	1.0	2	20	10	100
	5	2.7	0	20	0	95
	10	7.7	0	20	0	100
	20	16	0	20	0	100

* Percentage hatched based on surviving embryos.

The results obtained (based on mean measured concentrations of test substance) were

Time	LC50 (mg/L)
48 h	>16
96 h	>16

 

Based on mortality compared to the control (p <0.05) the 96 h No Observed Effect Concentration (NOEC) was determined to be 16 mg/L and the Lowest Observed Effect Concentration (LOEC) was >16 mg/L. There was no mortality observed in the internal plate controls. The control had 4% mortality which is acceptable. The positive control had 100% mortality at 96 h.

 

Validity criteria

The OECD 236 Guideline details the following performance criteria for the test validity:

- The overall fertilisation rate of all eggs collected should be ≥70% in the batch tested.

- The water temperature should be maintained at 26 ± 1 deg C in the test chambers at any time during the test.

- Overall survival of embryos in the dilution water control and where relevant, in the solvent control should be ≥90% until the end of the 96 h exposure.

- Exposure to the positive control (4 mg/L 3,4-dichloroaniline) should result in a minimum mortality of 30% at the end of the 96 h exposure.

- Hatching rate in the dilution water control and solvent control if appropriate, should be ≥80% at the end of the 96 h exposure.

- At the end of the 96 h, the dissolved oxygen concentration in the dilution water control and the highest test concentration should be ≥80% of saturation.

All validity criteria were met during the study.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the test substance 96 h LC50 and NOEC were determined to be >16 mg/L and 16 mg/L respectively.

Executive summary:

A study was conducted to determine the acute toxicity potential of the test substance, ‘mono- and di- C16-18 PSE and C16 -18 AE10 PSE' to Zebrafish (Danio rerio), according to OECD Guideline 236 (Fish Embryo Toxicity (FET)), in compliance with GLP. The test was initiated by the addition of 1 impartially selected Zebrafish embryo, to each 24 well. The embryos were pre-exposed in petri dishes and sorted prior to addition to the well plates within 90 minutes of fertilisation. Fertilised eggs, undergoing cleavage and showing no obvious irregularities during cleavage or injuries of the chorion were selected. Loading of embryos to the well plates was completed less than 3 h post fertilisation. The test organism were exposed to dilution water control and positive control (3,4-dichloroaniline) together with nominal test substance exposure concentrations of 1.25, 2.5, 5, 10 and 20 mg/L. Levels were based on the dispersibility observed in a non-GLP solubility trial and range finding test. Analytical dose verification was conducted via HLPC. The mean measured concentrations of the test substance were determined to be 0.24, 1, 2.7, 7.7 and 16 mg/L. An assessment of the response of the Zebrafish embryos was made at 24, 48, 72 and 96 h after the commencement of the test using a low power binocular microscope, with bright field illumination. Any positive outcome of the four observations (coagulation of the embryo, lack of somite formation, non-detachment of the tail, lack of heartbeat) meant that the zebrafish embryo was dead. The 96 h and 48 h LC50 (median lethal concentration) were determined to be >16 mg/L (measured). Based on mortality compared to the solvent control (p <0.05) the 48 h NOEC was determined to be 16 mg/L (measured) and the LOEC was >16 mg/L (measured). All validity criteria were met during this study. Under the study conditions, the 96 h LC50 and NOEC values of the test substance were >16 mg/L and 16 mg/L respectively (Scymaris, 2018).

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From Janaury 14, 2013 to February 27, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

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)

Remarks:

Institut für Biologische Analytik und Consulting, IBACON GmbH, Arheilger Weg 17, 64380 Rossdorf, Germany

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken at the start of the test. For the determination of the stability of the test substance under the test conditions and of the maintenance of the test substance concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 96 h) from the approximate centre of the aquaria.

Vehicle:

no

Details on test solutions:

Preparation and application of test solution
- Method:
The test substance was not well soluble in test water. Therefore, a stock suspension of 100 mg test substance/L was prepared by suspending 1.0002 g of test substance in 10.0002 L test water for preparing the test concentration. The stock suspension was stirred for 24 h to dissolve as much test substance as possible. Then, the undissolved test substance was separated by filtration (0.45 µm cellulose acetate nitrate filter). The test media were prepared just before introduction of the test fish (= start of the test).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test organism
- Common name: Rainbow trout
- Source: The test fish were obtained from Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany
- Age at study initiation: Juveniles
- Length at study initiation: 4.83 cm ± 0.30 cm
- Weight at study initiation: 1.12 ± 0.23 g
- Feeding during test: no

Acclimation
- Acclimation period: All fish were obtained and held in the laboratory for at least 12 d before the start of the test.
- Acclimation conditions: same as test conditions
- Feeding frequency: three times per week or daily until 24 h before the test was started
- Health during acclimation: During the last 7 d prior to the start of the test no fish died in the test fish batch. Therefore the mortalities in the fish batch were below 5 % and the fish batch was accepted.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

2.5 mmol/L (= 250.0 mg/L) as CaCO3

Test temperature:

15°C

pH:

7.8 to 8.2

Dissolved oxygen:

93 to 98 % of the air saturation value

Nominal and measured concentrations:

A filtrate of a supersaturated stock suspension of nominal 100 mg/L. No measured concentrations are available (below LOD).

Details on test conditions:

Test system
- Test vessel: 12 L glass aquaria with 10 L test medium
- Aeration: The test media were slightly aerated during the test.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1

Test medium / water parameters
- Chlorine: 2.0 mmol/L (= 294.0 mg/L)
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4: 1 (based on molarity)
- Conductivity: < 10 µScm-1
- Culture medium different from test medium: no

Other test conditions
- Adjustment of pH:
- Photoperiod: 16 h light: 8 h dark; 30 min dawn/dusk period was provided
- Light intensity: 780 to 810 lux

Test concentrations
- Test concentrations:
The test substance was not well soluble in test water. Therefore, a stock suspension of 100 mg test substance/L was prepared by suspending 1.0002 g of test substance in 10.0002 L test water for preparing the test concentration. The stock suspension was stirred for 24 h to dissolve as much test substance as possible. Then, the undissolved test substance was separated by filtration (0.45 µm cellulose acetate nitrate filter). The test media were prepared just before introduction of the test fish (= start of the test).

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: up to the water solubility of the test substance no mortality of the test animals occurred.

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: up to the water solubility of the test substance no mortality of the test animals occurred.

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

Results and discussion

Validity criteria of the study

Control: In the control no fish died until the end of the test.

Dissolved Oxygen Concentration: The dissolved oxygen concentration in the test media did not fall below 93 % of air saturation value during the test.

 

Biological results

Sublethal Effects: In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.

96 h LC50: > 100 mg test substance/L (nominal)

95 % Confidence Interval: Not determinable

96 h LC10: > 100 mg test substance/L (nominal)

95 % Confidence Interval: Not determinable

96 h LC0: 100 mg test substance/L (nominal)

96 h LC100: > 100 mg test substance/L (nominal)

96 h NOEC: 100 mg test substance/L (nominal)

96 h LOEC: > 100 mg test substance/L (nominal)

Table: Mortality and sublethal effects

Exposure time (h)	Effects	Nominal concentration (mg/L)
		Control	100
0 h	#Mortality	0	0
	#Sublethal effects	0	0
2 h	#Mortality	0	0
	#Sublethal effects	0	0
24 h	#Mortality	0	0
	#Sublethal effects	0	0
48 h	#Mortality	0	0
	#Sublethal effects	0	0
72 h	#Mortality	0	0
	#Sublethal effects	0	0
96 h	#Mortality	0	0
	#Sublethal effects	0	0

Analytical Results

Determination of the test substance: Based on the results of LC-MS/MS measurements the concentration of the test substance was determined using a calibration curve.

Calibration range: 0.005 to 0.15 mg test substance/L

Linearity of response: Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression

Regression coefficient (r2): 0.9994

Calibration curve: y = 12112 * x + 10276 (see also Figure 1)

Limit of detection: 1.6 μg test substance/L

Limit of quantification: 18.83 μg test substance/L

The Limit of quantification was calculated according to DIN 32645 from the calibration curve. A preparation of fortified samples was not possible, since the test substance is not soluble in the test medium of the aquatic test.

Mean Recovery in the Fortified

Samples: 60 % (n = 10, RSD 16 %)

The measurements showed unsatisfying recoveries. This might be caused by the bad solubility of the test substance in test medium of the aquatic test.

As the solubility of the test substance in the test medium is low, the concentrations of dissolved test substance were below the limit of detection. However, all reported results refer to nominal concentrations.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 96 h LC50 and NOEC for the test substance, can be considered to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively.

Executive summary:

A study was conducted to determine the toxic potential of read across substance, mono- and di- C16 PSE, K+ (purity: ca. 85%) to rainbow trout (Oncorhynchus mykiss), according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Seven juvenile fish were exposed to the test substance at nominal loading rate of 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for sublethal effects and mortality. As the test substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. The quantification of the test substance was performed in duplicates using liquid chromatography (LC-MS/MS-method). The concentrations of dissolved test substance were found to be below the Limit of Detection (1.6 μg test substance/L). Therefore, all results were presented in terms of nominal loading rates. In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC for the read across substance were determined to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively (Kuhl and Wydra, 2013). Based on the results of the read across study, similar 96 h LC50 and NOEC values can be expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE PSE (10EO)'.

Description of key information

Based on the results of the available studies, no toxicity to fish is expected up to the maximum soluble concentrations of the test substance. The lower LC50 value of 16 mg/L (nominal), based on the FET study with the test substance has been considered further for the hazard assessment, as a conservative approach.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

16 mg/L

Additional information

For the short-term toxicity to fish endpoint, only a fish embryo toxicity (OECD TG 236) test is available with the test substance, 'mono- and di- C16 -18 PSE and C16 -18 AE10 PSE'. The FET study according to the official ECHA recommendations (ECHA report, 2016), is to be used within a weight of evidence approach (Annex XI, Section 1.2 to the REACH Regulation) together with other independent, adequate, relevant and reliable sources of information leading to the conclusion that the substance has or does not have a particular dangerous property. Therefore, the FET of the test substance is supported with the study available on a substance representative of one of the main constituent, which is alcohol phosphates (PSE). No further testing or read across is required for the test substance, as based on the available data, fish has not been identified as the most sensitive species.

FET study with the test substance

A study was conducted to determine the acute toxicity potential of the test substance, ‘mono- and di- C16-18 PSE and C16 -18 AE10 PSE' to Zebrafish (Danio rerio), according to OECD Guideline 236 (Fish Embryo Toxicity (FET)), in compliance with GLP.The test was initiated by the addition of 1 impartially selected Zebrafish embryo, to each 24 well. The embryos were pre-exposed in petri dishes and sorted prior to addition to the well plates within 90 minutes of fertilisation. Fertilised eggs, undergoing cleavage and showing no obvious irregularities during cleavage or injuries of the chorion were selected. Loading of embryos to the well plates was completed less than 3 h post fertilisation. The test organism was exposed to dilution water control and positive control (3,4-dichloroaniline) together with nominal test substance exposure concentrations of 1.25, 2.5, 5, 10 and 20 mg/L. Levels were based on the dispersibility observed in a non-GLP solubility trial and range finding test.Analytical dose verification was conducted via HLPC.The mean measured concentrations of the test substance were determined to be 0.24, 1, 2.7, 7.7 and 16 mg/L. An assessment of the response of the Zebrafish embryos was made at 24, 48, 72 and 96 h after the commencement of the test using a low power binocular microscope, with bright field illumination. Any positive outcome of the four observations (coagulation of the embryo, lack of somite formation, non-detachment of the tail, lack of heartbeat)meant that the zebrafish embryo was dead. The 96 h and 48 h LC50 (median lethal concentration) were determined to be >16 mg/L (measured). Based on mortality compared to the solvent control (p <0.05) the 48 h NOEC was determined to be 16 mg/L (measured) and the LOEC was >16 mg/L (measured). All validity criteria were met during this study. Under the study conditions, the 96 h LC50 and NOEC values of the test substance were >16 mg/L and 16 mg/L respectively (Scymaris, 2018).

Constituent: PSE - read across study

A study was conducted to determine the toxic potential of read across substance, mono- and di- C16 PSE, K+ (purity: ca. 85%) to rainbow trout (Oncorhynchus mykiss), according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Seven juvenile fish were exposed to the test substance at nominal loading rate of 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for sublethal effects and mortality. As the test substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. The quantification of the test substance was performed in duplicates using liquid chromatography (LC-MS/MS-method). The concentrations of dissolved test substance were found to be below the Limit of Detection (1.6 μg test substance/L). Therefore, all results were presented in terms of nominal loading rates. In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC for the read across substance were determined to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively (Kuhl and Wydra, 2013). Based on the results of the read across study, similar 96 h LC50 and NOEC values can be expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE'.

Overall based on the available information, no toxicity to fish is expected upon exposure to the test substance. However, the lower LC50 value of 16 mg/L (nominal) has been considered further for the hazard assessment, as a conservative approach.