Saccharomyces cerevisiae cell wall, extracted

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 14, 2011 to January 24, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no analytical control and the methodology of exposure is not the WAF as recommended in OECD 23

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

(April 2004)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

The substance is a very complex UVCB.

Details on sampling:

No sampling for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions have been prepared from a stock solution of the test item previously diluted in Daphnids dilution water and by using ultra-sounds (10 min.) The stock solution was continuously stirred during the preparation of the test solutions.
- Controls: Only the mineral medium (without the tested item)
- Positive control : K2Cr2O7
- Chemical name of vehicle : no vehicule used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): yes. A slight deposit was noted after 48h in the preliminary test at the concentration of 100 mg/L. An important deposit was noted at the bottom of each container at the concentrations 800, 1600 and 3200 mg/L.
- Other relevant information:
- In the preliminary test : 3 tested concentrations at 1, 10 and 100 mg/L, under static conditions, for 48h. The tested solutions and the control (mineral solution) were added in two containers containing 5 daphnids each.
- in the final test : 6 tested concentrations at 100, 200, 400, 800, 1600, and 3200 mg/L, under semi-static conditions, for 48h. The tested solutions and the control (mineral solution) were added in two containers containing 5 daphnids each.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: clone number 5
- Age at study initiation (mean and range, SD): less than 24 h
- Length at study initiation (length definition, mean, range and SD): between 560 µm and 800 µm (obtained by filtration)

- Method of breeding: in closed bottles placed in a climatic chamber free from any toxic vapour
- Age of parental stock (mean and range, SD): From at least the 3rd génération (non cyclic parthenogenesis reproduction)
- Feeding during test : no
- Food type: no information

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

250 mg/L CacO3

Test temperature:

19.9- 20.2°C

pH:

At T0 pH between 7.8 - 6.5 and the pH decreases when the concentration of the test item increases.
At 48h pH between 8 - 6.7 and the pH decreases when the concentration of the test item increases.

Dissolved oxygen:

At T0 the oxygen concentration is between 9.3 - 8.4 mg/L
At 48h the oxygen concentration is between 9.3 - 1.7 mg/L and the oxygen decreases quickly when the concentration of the test item increases. For the concentrations of 400 mg/L or higher the oxygen concentrations are < 3 mg/L.

Salinity:

Not relevant

Conductivity:

not reported

Nominal and measured concentrations:

The following nominal concentrations were tested in the final test : 100; 200; 400; 800; 1800; 3200 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 containers per concentration
- Type (delete if not applicable): covered
- Volume of solution: 10 ml of medium
- Aeration: no
- Renewal rate of test solution : every 24 day
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: mineral medium as described in OECD 202 guideline (Cacl2, 2H2O : 297 mg/L; MgCl2,6H2O: 167 mg/L; NaHCO3: 200 mg/L; K2SO4:26 mg/L)
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: 7.8
- Ca/mg ratio: not reported
- Conductivity: not reported
- Culture medium different from test medium: not reported
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH not reported
- Photoperiod: darkwithin 15 sec. after a gentle agitation of the containers)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observation of Daphnids mobility made after 24h and 48h of exposition (Immobilisation = Inability for the daphnids to swim within 15 sec. after a gentle agitation of the containers).

RANGE-FINDING STUDY
- Test concentrations: 1; 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No immobilisation at the 3 tested concentrations

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

morphology

Key result

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Details on results:

Due to a important decrease of the oxygen concentration measured at 48h (less than 3 mg/L), the following tested concentrations were not taken into account : 400; 800; 1600 and 3600 mg/L.
An important deposit was observed at the bottom of each container at 800, 1600 and 3200 mg/L.
No signs of stress was observed on the mobile daphnids after 48hours of exposure.

Results with reference substance (positive control):

EC50-24 h = 1.18 mg/L (1.08-1.29 mg/L) within the acceptable interval of 0.6-2.1 mg/L. The daphnids sensitivity is correct.

Daphnids immobilization after 24h and 48h of exposure to the test item

Nominal Test Item Concentration (mg/L)	Immobilised Daphnids at 24h	Immobilised Daphnids at 24h
0	0	0
100	0	0
200	0	1 (5%)
400	0	7 (35%)
800	2 (10%)	12 (60%)
1600	12 (60%)	20 (100%)
3200	8 (40%)	20 (100%)

 

 

pH and dissolved oxygen values at the start and the end of the final test

Nominal Test Item Conc. (mg/L)	pH		Dissolved oxygen in mg/L
	T0	T48h	T0	T48h
0	7.8	8	9.3	9.3
100	7.8	7.8	9.2	7.4
200	7.8	7.4	9.1	5.2
400	7.7	7.3	9.0	2.7
800	7.2	7.3	9.0	1.9
1600	6.9	7.0	8.8	2.3
3200	6.5	6.7	8.4	1.7

 

Validity criteria fulfilled:

yes

Remarks:

The validity criteria are fullfilled with less than 10% immobilisation in the control and an oxygen concentration > 3 mg/L for the tested concentrations of 100 and 200 mg/L.

Conclusions:

No significant toxic effect was observed at 100 and 200 mg/L. So the EL50-48h is higher than 200 mg/L.

Executive summary:

The acute toxicity of "Saccharomyces cerevisiae cell wall, extracted", to Daphnia magna, was determined according to the OECD 202 Guideline and in compliance with the GLP.

4 testing containers containing each 5 daphnids were exposed for 48h, under semi-static conditions, to the following test concentrations: 100; 200; 400; 800; 1600 and 3200 mg/L. The tested solutions were prepared from a stock solution of the test item, previously diluted in the Daphnids mineral medium, and then treated with ultra-sounds (10 min.) to removed the heads. The stock solution was continously stirred during the preparation of the test solutions. Althought this procedure is not the recommended methodology for insoluble UVCB, the saturation is likely reached for each ingredient of this UVCB, due to the very high concentrations tested.

Moreover it is indicated by the study director that the WAF (which is the suitable method for insoluble UVCB) cannot apply due to the size of the particles (however this explanation is not clear).

No analysis was performed on the tested solutions due to the complex composition of this UVCB.

No mortality was observed in the control. An important decrease of the oxygen concentration was observed at 48h (less than 3 mg/L), and the corresponding tested concentrations were not taken into account. Only the tested concentrations 100 and 200 mg/L for which the oxygen concentration was > 3 mg/L were considered for the toxicity determination with 0% and 5% immobilisation of the Daphnids, respectively.

No signs of stress was observed on the mobile daphnids after 48hours of exposure.

The 48h-EL50 to Daphnia magna of "Saccharomyces cerevisiae cell wall, extracted" is > 200 mg/L and the 48h-EL0 is 100 mg/L.

This acute toxicity test to Daphnia magna is classified as Klimish 2 because of the choice of the methodology and the lack of analytical control. Nevertheless it was demonstrated that "Saccharomyces cerevisiae cell wall, extracted" does not show any toxicity to Daphnids.

 

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Study period:

29 November 2017 to 14 March 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Justification for type of information:

See the attached document for the justification concerning the use of the acute Daphnia test on Saccharomyces cerevisiae lysate, as a supporting study, which reinforces the statement that the substance "Saccharomyces cerevisiae cell wall, extracted" shows no adverse effect on Daphnia magna, based on a Key study.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 13 April 2004

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)

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.

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 (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

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

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

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 checked 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 checked 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.

 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

 

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 48h of exposure to the loading rate of 100 mg/L test item (WAF solution). The 48h-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.

Description of key information

The acute toxicity of "Saccharomyces cerevisiae cell wall, extracted" to Daphnia magna, was determined according to the OECD 202 Guideline and in compliance with the GLP.

At the concentrations of 100 mg/L and 200 mg/L, the daphnids immobilisation was 0% and 5 % respectively. No analysis was performed on the tested solutions due to the complex composition of this UVCB. The 48h-EL50 to Daphnia magna of "Saccharomyces cerevisiae cell wall, extracted" is > 200 mg/L and the 48h-EL0 is 100 mg/L.

Saccharomyces cerevisiae cell wall, extracted, does not show any toxicity to Daphnids.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

200 mg/L

Additional information

This lack of toxicity of "Saccharomyces cerevisiae cell wall, extracted" is reinforced by a supporting study performed on a close substance "Saccharomyces cerevisiae lysate" which also demonstrated no adverse effect to Daphnia magna.