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Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2017 to 14 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP

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 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
EC No.1907/2006
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 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 into the tes medium (ISO media). The highest carbon content was obtained after 0.5 hour of stirring of a WAF of 100 mg/L test item at 18-22°C. 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 (ISO medium) before the start of the experiment. These solutions were shaken for approximately 0.5h hour continuously at the test temperature (18-22°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 vessels prior to introduction of Daphnia. 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 chromosulfuruc 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 vessels prior to introduction of Daphnia.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnids
- Strain/clone: Daphnia magna
- Justification for species other than prescribed by test guideline: Daphnia magna is the standard species of the acute immobilisation test.
- Source: Szent István University, Department of Aquaculture 2100 Gödöllő, Páter Károly u. 1. – Hungary
- Age of tested animals: less than 24 h old at the beginning of the test
- Feeding during test : no

ACCLIMATION
- Acclimation period: There was no acclimatization because the water used was similar to the culture water
- Type and amount of food: not reported
- Feeding frequency: not reported
- Health during acclimation (any mortality observed): not reported

Study design

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

Test conditions

Hardness:
246 mg/L (as CaCO3).
Test temperature:
20.1 – 20.6°C measured in the test vessels.
pH:
7.20 – 7.49
Dissolved oxygen:
6.9 – 8.9 mg/L
Salinity:
Not appropiate
Conductivity:
Not reported
Nominal and measured concentrations:

Tested nominal concentration : 100 mg/L test item
Measured concentration : 15.9 mg/L carbon
The test item is an UVCB, with a very complex composition and there is 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 daphnids 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 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: glass beaker
- Material, size, headspace, fill volume: 5ml/test animal
- Aeration: no
- Renewal rate of test solution : every 24h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: desionised water
Stock solutions Amount added Stock solutions
(g/L) (ml/L)
CaCl2×2 H2O 11.76 25
MgSO4×7 H2O 4.93 25
KCl 0.23 25
NaHCO3 2.59 25

- Culture medium different from test medium: no
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light and 8-hour dark cycle.
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 0.1; 1; 10; 100 mg/L
- Results used to determine the conditions for the definitive study: No toxicity was observed in the preliminary study therefore 100 mg/L Test Item nominal loading rate (WAF) and one Control was used in the main test based on the results of the preliminary rang-finding test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mobility
Remarks on result:
other: the tested concentration was cheched through the NPOC measurements. A stable concentration of 15.9 ± 0.6 mg/L carbon was measured during the test.
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mobility
Remarks on result:
other: the tested concentration was cheched through the NPOC measurements. A stable concentration of 15.9 ± 0.6 mg/L carbon was measured during the test.
Details on results:

No immobilisation was observed in the treated vessels during the study.
There was no immobilized animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits.
Results with reference substance (positive control):
Potassium dichromate is tested at least twice a year (last control 06-07 December 2017). The 24h EC50: 0.65 mg/L, (95% confidence limits: 0.61–0.70 mg/L).
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects.

Any other information on results incl. tables

 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

15.2 ± 5.21

15.3 ± 3.06

Second renewal period

16.5 ± 1.39

16.4 ± 1.00

 

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

14 March 2018

-16.6

2.88

0.9989

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 acute toxicity of Saccharomyces cerevisiae lysate to Daphnia magna was studied and no adverse effect was observed during the 48 of exposure to the loading rate of 100 mg/L test item (WAF solution).
The 96h-EL50 > 100 mg/L test item (WAF solution) and the NOELR is equal to 100 mg/L (WAF solution) which corresponds to 15.9 mg/L carbon content.
Executive summary:

The acute toxicity of Saccharomyces cerevisiae lysate to Daphnia magna was determined according to EU Method C.2 and OECD 202, and in compliance with GLP.

20 daphnids distributed in 4 vessels were exposed to the test item in a semi-static test during 48 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 0.5h 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 there 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 renewal periods (from an additional vessel without the test organisms which would affect the total carbon present in solution). The mean measured concentration was 15.9 ± 0.6 mg carbon /L and was stable during the exposure periods.

No immobilisation was observed during the study at the loading rate of 100 mg/L test item (WAF solution). There was no immobilized animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits.

The 48h-EL50 to Daphnia magna of Saccharomyces cerevisaie lysate is superior to 100 mg/L (WAF solution) and the NOELR is equal to 100 mg/L (WAF solution), which corresponds to 15.9 mg/L carbon content.

This acute toxicity to Daphnia magna is classified as acceptable, and satisfies the guideline requirements of EU Method C.2 and OECD 202.