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Short-term toxicity to fish

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Reference
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. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
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
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 aclohol 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 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).

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.