Registration Dossier

Diss Factsheets

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 29 November 2017 to 16 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
Adopted July 17, 1992
Deviations:
yes
Remarks:
The recovery of test item from the media was not possible due to the characteristic of test item, therefore the carbon content of soluble fractions was determined.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Saccharomyces cerevisiae, lysate
EC Number:
305-230-8
EC Name:
Saccharomyces cerevisiae, lysate
Cas Number:
94350-12-6
IUPAC Name:
Saccharomyces cerevisiae, lysate

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The organic carbon content of Saccharomyces cerevisiae, lysate in the test solutions was determined at the beginning and at the end of the first and last renewal periods.
Samples were taken from the test solutions and the control solutions. The samples were filled into three different tubes for the triplicate measurements and they were measured by the NPOC method without any dilution, based on the method validation (Study code: 17/230-316AN). The measured carbon contents of the test solutions were corrected with the measured values of control samples.

Test solutions

Vehicle:
no
Details on test solutions:

Because the Test Item is a UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials), a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.

In a preliminary formulation study, the optimal conditions for the preparation of the water accomodated fraction (WAF) were determined. The highest carbon content at 20°C was obtained after 1 hour of stirring of a WAF of 100 mg/L test item. These conditions were applied for the preliminary range-finding study and the main test.

Four test concentrations in a geometric series (separation factor of 10) plus a control were used in the Preliminary range-finding test. The saturated Test Item solutions at 0.1, 1.0, 10, 100 mg/L nominal loading rates (WAFs) were prepared individually by dispersing/dissolving the needed amount of Test Item into the test medium (aquarium water) before the start of the experiment. These solutions were shaken for approximately 1 hour continuously around 20*C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solutions (conditions determined in the preliminary formulation study). The test formulations were prepared individually and distributed into test aquariums prior to introduction of fish. In order to reduce issues of biodegradation of the Test Item, all the glassware used to prepare the WAFs and test solutions were washed with 15-17% hydrochloric acid before use. The prepared solutions were tested immediately after their preparation.

Since no toxiciy was observed in the preliminary study, therefore 100 mg/L test item nominal loading rate (WAF) and one control was used for the main test. In the Main study, the test formulations were prepared individually as described above and distributed into test aquarium prior to introduction of fish.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Strain: Brachydanio rerio
- Source: Szent István University, Department of Aquaculture 2100 Gödöllő, Páter Károly u. 1. – Hungary
- Age at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD): 2.5 – 2.8 cm
- Weight at study initiation (mean and range, SD): mean weight in control = 0.15 g/fish; mean weight in 100mg/L = 0.0.21 g/fish
- Maintenance of the brood fish: 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.

ACCLIMATION
- Acclimation period: > 12 days
- Acclimation conditions (same as test or not): not reported
- Type and amount of food during acclimation: not reported
- Feeding frequency during acclimation: not reported
- Health during acclimation (any mortality observed): Mortality was less than 5% of the population for seven days before the test.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h

Test conditions

Hardness:
189 mg/L (as CaCO3)
Test temperature:
20.3–21.3°C
pH:
7.24–7.73
Dissolved oxygen:
78–96 % of the air saturation value at the temperature used
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:

Tested nominal concentration : 100 mg/L test item
Measured concentration : 17.5 mg/L carbon
The test item is an UVCB, with a very complex composition and no single chemical marker that can be readily analysed. Therefore a global analytical method, the non-purgeable organic carbon (NPOC) method was used to demonstrate that the fish were exposed to the maximum soluble concentration and that this concentration was stable during the test.
The concentration of the Test Item was measured in the test solution (also in the Control) at start and at the end of the first and last renewal periods (from an additional vessel without the test organism which would affect the total carbon present in solution).
Details on test conditions:

TEST SYSTEM
- Test vessel: 1 aquarium
- Material, size, headspace, fill volume: 5 L test solution
- Aeration: not reported
- Renewal rate of test solution : every 24 h (semi-static)
- No. of organisms per vessel: 7 fish
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: < 1g fish/L

TEST MEDIUM / WATER PARAMETERS : not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality or any sub-lethal effect was observed at 3, 6, 24, 48, 72 and 96 hours.
The test conditions (pH, temperature, oxygen saturation) were examined at the start and at the end of the renewal periods.
The body weights of the fish were recorded before the introduction of fish and the loading rate was calculated.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Range finding study : 1.01; 1; 10 and 100 mg/L test item loading rate (WAF)
- Test concentrations: 100 mg/L test item loading rate (WAF)
- Results used to determine the conditions for the definitive study: no mortality in the range finding study.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mortality (fish)
Remarks on result:
other: the tested concentration was cheched through the NPOC measurements. A stable concentration of 17.5 mg/L carbon was measured during the test.
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mortality (fish)
Remarks on result:
other: the tested concentration was cheched through the NPOC measurements. A stable concentration of 17.5 mg/L carbon was measured during the test.
Details on results:
No mortality or any sub-lethal effect was observed during the study.
Constant conditions were maintained in this study’s procedure.
No mortality occurred in the control (validity criteria ≤ 10 % at the end of the test).
The dissolved oxygen concentration (throughout the test) was at least 60 % of air saturation value.
The following validity criteria: “The concentration of the Test Item should be at least 80 % of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20%, results will be based on the measured concentration.” was not applicable as only the sum of the carbon content of the Test Item could be measured by NPOC, and by this it was justified that the Test Item was present in the test solutions and is maintained during exposure periods.
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects.

Any other information on results incl. tables

Sublethal observations / clinical signs:

ANALYSIS OF CARBON CONTENT

Measured carbon contents of the renewal periods (24 h)

Nominal conc. 100 mg/L Test Item (WAF)

Measured carbon content

at the start

Measured carbon content at the end

mg/L

mg/L

First renewal period

17.4 ± 4.72

17.8 ± 1.58

Fourth renewal period

17.4 ± 1.08

17.2 ± 0.483

 

Data of the regression lines

Date of measurement

Intercept

Slope

Correlation Coefficient

12 March 2018

-8.21

2.91

0.9999

13 March 2018

-12.9

2.87

0.9998

15 March 2018

-13.8

2.76

0.9998

16 March 2018

-11.9

2.81

0.9998


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The control of the tested concentration (WAF at 100 mg/L) was performed through the NPOC measurements.
Conclusions:
The toxicity of Saccharomyces cerevisiae lysate to fish was determined in an acute toxicity test on Brachydanio rerio, and no adverse effect was observed during the 96 hours of exposure to the loading rate of 100 mg/L test item (WAF solution).
The 96h-LL50 > 100 mg/L test item (WAF solution) and the NOELR is equal to 100 mg/L (WAF solution) which corresponds to 17.5 mg/L carbon content.
Executive summary:

The acute toxicity of Saccharomyces cerevisiae lysate to fish was determined according to EU method C.1 and OECD 203, and in compliance with GLP.

Brachydanio rerio was exposed to the test item in a semi-static test during 96 hours. In a preliminary formulation study, the optimal conditions for the preparation of the Water Accomodated Fraction (WAF) were determined. The highest carbon content at 20°C was obtained after 1 hour of stirring of a 100 mg/L test item, loading rate followed by filtration through a fine (0.22 µm) filter to give the 100% saturated solutions (WAF). Since no toxicity was observed during the range-finding study, a limit test at the loading rate of 100 mg/L test item (WAF solution) was performed.

Since the test item is an UVCB, with a very complex composition and ther is no single chemical marker that can be readily analysed, the non-purgeable organic carbon (NPOC) method was used to control the tested concentration. The concentration was measured at start and at the end of the first and last renewal periods (from an additional vessel without the test organism which would affect the total carbon present in solution). The mean measured concentration was 17.5 mg carbon /L and was stable during the exposure periods.

No mortality or any sub-lethal effect was observed during the study at the loading rate of 100 mg/L test item (WAF solution). No mortality occurred in the control and constant conditions were maintained during this test with temperature between 20.3–21.3°C, pH between 7.24–7.73 and dissolved oxygen ≥ 78%.

The 96h-LL50 to fish of Saccharomyces cerevisaie lysate is superior to 100 mg/L (WAF solution) and the NOELR is equal to 100 mg/L (WAF solution), which correspond to 17.5 mg/L carbon content.

This acute toxicity to fish is classified as acceptable, and satisfies the guideline requirements of EU Method C.1 and OECD 203.

Categories Display