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Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 - 13 Mar 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Measured concentrations were below the limit of quantification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

1996

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2017

GLP compliance:

yes (incl. QA statement)

Remarks:

National Institute of Pharmacy and Nutrition, Budapest, Hungary (date of issue: 03 August 2018)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L nominal loading rate WAF was analytically determined at the start and at the end of the first and at the last renewal periods.
- Sampling method: Four samples were taken (2 x 2 x ~15 mL) from the control and at the applied test concentration level at the beginning and at the end of the renewal periods
- Sample storage conditions before analysis: Samples were frozen and kept approximately at -20 °C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set was retained as a back-up at the Test Facility, if required for any confirmatory analyses.

Vehicle:

no

Details on test solutions:

- Method: The test item is poorly soluble in water, so test solutions were prepared using a saturated solution method (nominal loading rate of 100 mg/L, water accommodated fraction, WAF) according to the Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD No. 23. The saturated test item solution was prepared by dispersing/dissolving the amount of test item into the test medium (aquarium water) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30 °C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter. Prior to treatment of each renewal period, test item solutions were prepared by the method described above.
- Controls: yes, dilution water (circulated and filtered)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
- Length at study initiation (length definition, mean, range and SD): 2.4 – 2.7 cm
- Weight at study initiation (mean and range, SD): calculated mean weight of 1 fish = 0.25 - 0.27 g
- Method of breeding: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

ACCLIMATION
- Acclimation period: more than 12 days

FEEDING DURING TEST
- Food type: No

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

186 mg/L (as CaCO3)

Test temperature:

21.0 - 22.5 °C

pH:

7.78 – 8.34

Dissolved oxygen:

72 – 100% of the air saturation value

Nominal and measured concentrations:

- Nominal concentration: 100 mg/L nominal loading rate WAF
- Measured concentration 0 h (new): < LOQ
- Measured concentration 72 h (new): < LOQ
- Measured concentration 24 h (old): < LOQ
- Measured concentration 96 h (old): < LOQ

Details on test conditions:

TEST SYSTEM
- Test vessel: aquarium
- Fill volume: 5 L
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Control = 0.36 g fish/L, 100 mg/L test concentration = 0.38 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: circulated and filtered water
- Intervals of water quality measurement: The water temperature, oxygen concentration and pH were measured at the beginning and at the end of the renewal periods during the experiment in each test aquarium.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality or any sub-lethal effects were observed at 3, 6, 24, 48, 72 and 96 hours.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0.1, 1, 10, 100 mg/L nominal loading rate WAF
- Results used to determine the conditions for the definitive study: No effects in range finding study, NOEC = 100 mg/L nominal loading rate WAF

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

based on nominal loading rate WAF

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

based on nominal loading rate WAF

Details on results:

- Behavioural abnormalities: No, there were no clinical signs observed in the fish at any of the concentrations during the experiment.
- Observations on body length and weight: There was no considerable difference observed concerning body weights between the groups.

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of toxic effects.

Sublethal observations / clinical signs:

Table 1: Calculation of exposure concentrations

Nominal Loading rate WAFs (mg/L)	Measured concentrations 1st renewal period (mg/L)		Measured concentrations 2nd renewal period (mg/L)
	start	end	start	end
Control	<LOQ*	<LOQ*	<LOQ*	<LOQ*
100.0	<LOQ*	<LOQ*	<LOQ*	<LOQ*

*0.038 mg/L sample concentration (counting with 50x enrichment and 1.05x dilution during the sample preparation)

 

Table 2: Cummulative mortality data of danio rerio

Nominal Loading rate WAF mg/L	Total number of animals	Cummulative mortality of fish
		3 h	6 h	24 h	48 h	72 h	96 h
Control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

 

Table 3: Measured and calculated data of bodyweight

Test Group	Measured weight of 7 fish (g)	Calculated mean weight of 1 fish (g)	Loading of testing aquarium (g fish/L testing liquid)
Control	1.79	0.26	0.36
100.0 mg/L nominal loading rate	1.90	0.27	0.38

 

Table 4: Body length of test fish at mortality or at the end of the test

Loading rate WAF (mg/L)	Body length [cm]
Nominal
Control	2.5	2.7	2.6	2.6	2.7	2.5	2.6
100.0	2.6	2.7	2.5	2.6	2.4	2.7	2.6

 

Table 5: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	0%	Yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	72 – 100% air saturation throughout the test	Yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	The results were based on nominal concentrations, measured concentrations were below the limit of quantitation in this study	No

 

Validity criteria fulfilled:

no

Remarks:

See Table 5 at "any other information on results incl. tables".

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Suspensions of the 300 mg test substance were prepared in 3 l drinking water. shaking machine 130 rpm at room temperature for 24 h.
After 24 h the shaking was stopped and the solutions were filtered through paper filters directly into the test vessles used for the test, pH of the test solutions was not justified. The nominal concentration used for the test was 100 mg/l.
- Controls: yes, negative control

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain: not applicable
- Source: Aquafarm Ryba, Zeven, Germany
- Age at study initiation (mean and range, SD): not mentioned
- Length at study initiation: approx. 2.4 cm
- Weight at study initiation: not mentioned
- Method of breeding: not mentioned
- Feeding during test: none

ACCLIMATION
- Acclimation period: not mentioned

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

not mentioned

Test temperature:

22 - 23.4 °C

pH:

control: 7.62 - 8.31
test substance: 7.74 - 8.24

Dissolved oxygen:

control: 8.0 - 8.3 mg O2/L
test substance: 7.9 - 8.3 mg O2/L

Salinity:

not mentioned

Nominal and measured concentrations:

control / 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 5 L capacity glass tanks
- Aeration: not mentioned
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: not mentioned

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted
- Total organic carbon: not mentioned
- Particulate matter: not mentioned
- Alkalinity: not mentioned
- Ca/mg ratio: not mentioned
- Conductivity: not mentioned
- Culture medium different from test medium: no
- Intervals of water quality measurement: not mentioned

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: day/night-cycle 16 h/8h
- Light intensity: not mentioned

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): 24, 48, 72 and 96 h the test solutions were examined for dead animals. The pH was not adjusted furthermore during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study: not performed

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: not observed
- Observations on body length and weight: no changes in length and body weights compared to the negative controls
- Other biological observations: none
- Mortality of control: 0%
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: test conducted at test concentration above solubility of test substance

Results with reference substance (positive control):

no reference substance tested

Reported statistics and error estimates:

no statisitcs performed

Sublethal observations / clinical signs:

Table: Mortality (cumulative data)

concentration (nominal)[mg/L]	Number of fish	Mortality in %
		24 h	48 h	72 h	96 h
0 (control)	7	0	0	0	0
100	7	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

Under the present test conditions no toxic effects caused by the test item were observed at a nominal concentration of 100 mg/L exposure medium in a limit test. None of the animals died.

Executive summary:

In a 96-h acute toxicity study according to OECD 203 and GLP, zebrafish (Danio rerio) were exposed to the test substance (CAS 91744-38-6, Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts) at concentrations of 0 and 100 mg/L nominal (limit test) under semi-static conditions. The 96-h LL₅₀based on mortality was > 100 mg/L.  No sublethal effects were observed. Based on the results of this study, the test substance would be classified as non-toxic to Danio rerio.

Although the test substance has a low water solubility and the actual test concentration was not verified, the study is classified as acceptable. In WAF procedure, suspensions of the 300 mg test substance were prepared in 3L drinking water and shaken at room temperature for 24 h. After 24 h the shaking was stopped and the solutions were filtered through paper filters directly into the test vessels used for the test. Therefore it can be concluded that the test substance is not toxic at a loading of 100 mg/L (NOELR = 100 mg/L).

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to analogue justification document provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Description of key information

Danio rerio: 96-hour LL50 > 100 mg/L nominal loading WAF (OECD 203, 1992)

Danio rerio: 96-hour LL50 >100 mg/L (OECD 203); read-across

Additional information

The acute toxicity of Glycerides, C16-18 (even numbered) mono- and di- and their citrates (EC 701-358-7) towards fish was investigated in a 96-h acute toxicity GLP study according to OECD 203 (1992). Zebrafish (Danio rerio) were exposed to the test substance at 0 and 100 mg/L nominal loading rate water accommodated fractions (WAF) (limit test) under semi-static conditions. The 96-h LL50 based on mortality was > 100 mg/L. No sublethal effects were observed. The test item concentration could not be analytically quantified during the whole experiment as all measured concentrations were below the Limit of Quantification (LOQ = 0.038 mg/L). Therefore, all results are expressed as the nominal loading rate WAF.

Additionally, to support the results of the study with the test item in a read-across approach to the structurally and chemically related source substances Glycerides, C16 - 18 (even numbered) mono-, di-, and tri-, hydrogenated, citrates, potassium salt (CAS 917440-38-6), a 96-h acute toxicity GLP study was performed according to OECD 203. Zebrafish (Danio rerio) were exposed to the test substance (CAS 91744-38-6, Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts) at concentrations of 0 and 100 mg/L nominal (limit test) under semi-static conditions. The 96-h LL50 based on mortality was > 100 mg/L. No sublethal effects were observed.

Based on the results of both studies, no acute effects up to the water solubility (129 µg/L) towards fish are expected upon exposure with the target substance Glycerides, C16 - 18, mono- and di-, citrates.