Evernia prunastri, ext.

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:

2017-10-09 to 2017-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to the OECD 202 guideline. All validity criteria were successful.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

14 January 2016

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

In order to verify the maintenance of the WAFs during the ecotoxicological testing, chemical analyses in daphnids test solutions were performed:
- In fresh solutions at the beginning of the test (T = 0h) and after renewal (T=24h)
- In old solutions (T = 24h and T= 48h)
The test tube were pooled randomly (2*10mL) for chemical analyses.

Schedule analysis at initial time:
- T0h Control Fresh
- T0h 100.0 mg/L Fresh
All the “Fresh” solutions were analysed taken directly from the starting solutions without daphnids.

Schedule analysis at 24h:
- T24h Control Old
- T24h 100.0 mg/L Old

- T24h Control Fresh
- T24h 100.0 mg/L Fresh

Schedule analysis at final time:
- T48h Control Old
- T48h 100.0 mg/L Old

Vehicle:

no

Details on test solutions:

TEST ITEM PREPARATION
The test item was not soluble in the test medium, a special solubilisation protocol was carried out before the experimentation, according to the OECD n°23 guidance document on aquatic toxicity testing of difficult substances and mixtures.
After having tried different methods for the preparation of the WAFs in the preliminary study, the following protocol was selected for the final test: this method consisted in stirring the test item nominal concentration 100 mg/L in test medium during 24 hours, in the dark and 20 ± 2°C. The test solution (100 mg/L) was then filtered (cellulose acetate with a mesh size of 0.45 µm) to remove suspended particles: solution obtained was named WAF (Water Accommodated Fraction).
The test was carried out under semi static conditions, which means that the WAF solutions were renewed at 24h.

TEST SOLUTIONS
According to the results of the preliminary test, this study was conducted at one concentration level: 100.0 mg/L: Limit test, WAF and semi static conditions
The test contained a minimum of 12 glass test tubes as follow:
- Control: 4 replicates with daphnids (and 2 test tubes without daphnids for chemical analysis at initial time)
- WAF 100.0 mg/L: 4 replicates with daphnids (and 2 test tubes without daphnids for chemical analysis at initial time)

Each glass test tube (sterilized by autoclave) contained 10 mL of test solution and 5 daphnids; they were covered in order to avoid evaporation and dust deposition. Some abiotic solutions (without daphnids) were stored in the same conditions (test conditions) for analyses, if needed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Daphnia magna Straus, clone number 5, from at least the third generation (i.e. the third brood) obtained with a non-cyclic parthenogenesis reproduction.
The daphnids breeding was carried out in closed bottles placed in a climatic chamber free from any toxic vapour and in medium M4.
Daphnids used in this experimentation were previously filtered in order to obtain animals from a size between 560 µm and 800 µm (corresponding to daphnids less than 24 hours old.
- Feeding during test: None

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

260 - 270 mg/L of CaCO3

Test temperature:

19.8 - 20.0°C

pH:

7.8 - 7.9

Dissolved oxygen:

9.3 mg/L

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: limit test at WAF 100 mg/L (loading rate)

Details on test conditions:

TEST DETAILS
- Test vessel: glass test tube
- Type: closed
- Renewal rate of test solution: Renewal of test solutions at 24h
- No. of organisms per vessel: 5
- Test medium: The medium M4 ELENDT was prepared from concentrated stock solutions in ultrapure water as described in the guideline.
- Light intensity: Darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observations of the daphnid’s immobility and behaviour were done after 24 and 48 hours in control and exposed groups.
- Any sign of stress observed on the daphnids after 48 hours of exposure to the test item were reported.
- pH and dissolved oxygen were measured before and after renewal of solutions (including the controls).

RANGE-FINDING STUDY (non GLP)
- Test concentrations: control, 1, 10 and 100 mg/L with the same method of WAF preparation than the definitive test.
- Another non-GLP preliminary test with solvent conditions was also performed. Acetone has been determined as the most suitable solvent to be used. For stock solution at 100 g test item /L acetone, 4 loading rates 1; 10; 50 and 100 mg/L were prepared before acute immobilisation test with daphnids. During this study, it has not been possible to solubilize the test item at the maximal solvent concentration recommended in the OECD 23 guideline (100 mg/L or 0.1 mL/L). The solvent concentrations used were above this limit. Therefore, this method cannot be selected for the final test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mobility

Details on results:

No sign of stress was observed on the mobile daphnids after 48 hours of exposure to the highest loading rate.

Results with reference substance (positive control):

The last toxic response obtained on the reference item K2Cr2O7 (September 21, 2017) indicates an EC50-24h of 1.34 mg/L (1.22 – 1.47), indicating that the daphnids sensitivity is correct (within the accepted interval of 0.6–2.1 mg/L fixed by the International Standard ISO 6341 December 2012 as mentioned in the OECD 202 Guideline) and in accordance with historical data obtained by the laboratory.

Reported statistics and error estimates:

N/A

Table 6.1.3/1: Daphnids immobilisation

Raw data (daphnids immobilisation in cumulative number for each observation time 24 and 48 hours) are presented hereafter:

Nominal test item loading rate  in mg/L	Number of daphnids exposed at the start	Immobilised daphnids (%)
		at 24h	at 48h
WAF Control	20	0 (0%)	0 (0%)
WAF 100.0	20	0 (0%)	0 (0%)

Table 6.1.3/2: Chemical analysis of daphnids solutions

The table below summarizes the results achieved and the variation between initial measurement and after 24 hours and after renewal at 24 hours and 48 hours of exposure.

Nominal test item loading rate	Renewal	Results of the determination of TOC analysis (mg/L)
		At initial time (Fresh solutions)	After 24 hours of exposure(Old solutions)	Variation(%)
WAF 100 mg/L	T0h to T24h	(1.50)* < 2	(1.71)* < 2	NA
	T24h to T48h	2.68	2.76	+ 3.0

*(for information)

NA: Not Applicable

Some measured values were lower than 2 mg/L (=LOQ) and are notified for information (in brackets). The slight increase after 24 hours of exposition was mainly due to the gaseous exchange.

 

Overall, even if the TOC analyses indicated that very few organic compounds were found in the WAF at 100 mg/L, the test item « OAK MOSS CONCRETE» is considered to be stable under test conditions because the determination of the TOC analysis did not vary more than ± 20 % between the start and the end of the exposure periods. Moreover, it should be noted that the study was carried out using WAFs of an UVCB substance made of several constituents with different stability and behaviours in aqueous solutions during testing. Therefore, based on the results of the TOC analysis, due to the complex nature of the WAF and since the test item was an UVCB substance, it is considered acceptable to base the results on the nominal test loading rates.

Table 6.1.3/3: Validity criteria conformity

	Values for OECD 202	Validity criteria for the test
Immobilised daphnids in the control	=< 10 %	Ok (0%)
Dissolved Oxygen concentration at the end of the test	>= 3 mg/L	Ok (8.9 mg/L)

All the validity criteria were successful.

Validity criteria fulfilled:

yes

Conclusions:

Under test conditions, the results obtained for the test item “OAK MOSS CONCRETE” on the toxicological endpoint “immobilisation” give an EL50-24h and an EL50-48h higher than the highest tested loading rate, i.e. 100 mg/L, based on nominal test item loading rate.

Executive summary:

The aim of this study was to determine the acute toxicity of the test item “OAK MOSS CONCRETE” on the daphnids Daphnia magna, according to the OECD guideline202. The daphnidswere exposed toWater Accommodated Fractions (WAFs) of the test item, following the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No.23).

 

Following the results of a preliminary study, a limit test at a nominal loading rate of 100 mg test item/L was performed, in order to demonstrate that the test item has no significant effect on the test organism at this test loading rate or that EL₅₀ at 24h and 48h is higher than 100 mg/L. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours.

 

In order to check the stability of the WAFs in the test solutions, concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). These non-specific chemical analyses were performed by Carbon Analyzer. An overview of the results is given in the table below:

Nominal test item loading rate	Renewal	Results of the determination of TOC analysis (mg/L)
		At initial time (Fresh solutions)	After 24 hours of exposure(Old solutions)	Variation(%)
WAF 100 mg/L	T0h to T24h	(1.50)* < 2	(1.71)* < 2	NA
	T24h to T48h	2.68	2.76	+ 3.0

 

*(for information)

NA: Not Applicable

 

Therefore, according to the results of the TOC analysis and since the study was carried out using WAFs of an UVCB substance, the results were based on the nominal test loading rates.

 

Validity criteria conformity:

	Values for OECD 202	Validity criteria for the test
Immobilised daphnids in control	<10%	Ok (0%)
Dissolved Oxygen concentration	>3 mg/L	Ok (8.9 mg/L)

 

 

Daphnids immobilisation

Nominal test item loading rate  in mg/L	Number of daphnids exposed at the start	Immobilised daphnids (%)
		at 24h	at 48h
WAF Control	20	0 (0%)	0 (0%)
WAF 100.0	20	0 (0%)	0 (0%)

 

Conclusion

All the validity criteria of the study were successful. The results are as follows:

- EL50-24h is higher than 100 mg/L

- EL50-48h is higher than 100 mg/L

These values are based on nominal test item “OAK MOSS CONCRETE” loading rate.

Under test conditions, no ecotoxicity was observed after 24 hours and 48 hours of exposure during the testing at 100 mg/L of the test item (loading).

 

Therefore, the results obtained for the test item “OAK MOSS CONCRETE” on the toxicological endpoint “immobilisation” give an EL₅₀-24h and an EL₅₀-48h higher than the highest tested loading rate, i.e. 100 mg/L, based on nominal test item loading rate.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A read-across from a study performed on the analogue substance Oakmoss concrete (Concrete obtained from lichen of Evernia prunastri (Parmeliaceae) by extraction with a mixture of polar and apolar solvents) has been performed.
The registered substance (target) and the source substance are natural extracts belonging to the group of Natural Complex Substances (NCS): UVCB sub-type 3, where the source is biological, and the process is refinement (ECHA Guidance on Identification and naming of substances under REACH, version 2.1 – May 2017, Section 4.3.1 and EFEO/IFRA Guidelines on substance identification and sameness of natural complex substances (NCS) under REACH and CLP, version of August 5, 2015).

The source substance Treemoss concrete (Concrete obtained from lichen of Evernia prunastri (Parmeliaceae) by extraction with a mixture of polar and apolar solvents) and the registered substance Treemoss absolute (Absolute obtained from lichen of Evernia prunastri (Parmeliaceae) by extraction with a mixture of polar and apolar solvents followed by an extraction with ethanol solvent) have the same botanical origin: lichen of Evernia prunastri (Parmeliaceae). They have the same related EC and CAS numbers. The registered substance is obtained from the source substance Oakmoss concrete. The source substance contains all the constituents available from the extraction of the registered substance. Overall, they both have the same main constituents in similar concentrations, based on their respective SIP. As a consequence, the registered substance is expected to have its composition, and then its ecotoxicological behaviour, covered by Oakmoss concrete data. Therefore, we consider as reliable the read across from Oakmoss concrete dossier to this dossier.

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

N/A

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mobility

Details on results:

No sign of stress was observed on the mobile daphnids after 48 hours of exposure to the highest loading rate.

Results with reference substance (positive control):

The last toxic response obtained on the reference item K2Cr2O7 (September 21, 2017) indicates an EC50-24h of 1.34 mg/L (1.22 – 1.47), indicating that the daphnids sensitivity is correct (within the accepted interval of 0.6–2.1 mg/L fixed by the International Standard ISO 6341 December 2012 as mentioned in the OECD 202 Guideline) and in accordance with historical data obtained by the laboratory.

Reported statistics and error estimates:

N/A

Table 6.1.3/1: Daphnids immobilisation

Raw data (daphnids immobilisation in cumulative number for each observation time 24 and 48 hours) are presented hereafter:

Nominal test item loading rate  in mg/L	Number of daphnids exposed at the start	Immobilised daphnids (%)
		at 24h	at 48h
WAF Control	20	0 (0%)	0 (0%)
WAF 100.0	20	0 (0%)	0 (0%)

Table 6.1.3/2: Chemical analysis of daphnids solutions

The table below summarizes the results achieved and the variation between initial measurement and after 24 hours and after renewal at 24 hours and 48 hours of exposure.

Nominal test item loading rate	Renewal	Results of the determination of TOC analysis (mg/L)
		At initial time (Fresh solutions)	After 24 hours of exposure(Old solutions)	Variation(%)
WAF 100 mg/L	T0h to T24h	(1.50)* < 2	(1.71)* < 2	NA
	T24h to T48h	2.68	2.76	+ 3.0

*(for information)

NA: Not Applicable

Some measured values were lower than 2 mg/L (=LOQ) and are notified for information (in brackets). The slight increase after 24 hours of exposition was mainly due to the gaseous exchange.

 

Overall, even if the TOC analyses indicated that very few organic compounds were found in the WAF at 100 mg/L, the test item « OAK MOSS CONCRETE» is considered to be stable under test conditions because the determination of the TOC analysis did not vary more than ± 20 % between the start and the end of the exposure periods. Moreover, it should be noted that the study was carried out using WAFs of an UVCB substance made of several constituents with different stability and behaviours in aqueous solutions during testing. Therefore, based on the results of the TOC analysis, due to the complex nature of the WAF and since the test item was an UVCB substance, it is considered acceptable to base the results on the nominal test loading rates.

Table 6.1.3/3: Validity criteria conformity

	Values for OECD 202	Validity criteria for the test
Immobilised daphnids in the control	=< 10 %	Ok (0%)
Dissolved Oxygen concentration at the end of the test	>= 3 mg/L	Ok (8.9 mg/L)

All the validity criteria were successful.

Validity criteria fulfilled:

yes

Conclusions:

Under test conditions, the results obtained for the test item “OAK MOSS CONCRETE” on the toxicological endpoint “immobilisation” give an EL50-24h and an EL50-48h higher than the highest tested loading rate, i.e. 100 mg/L, based on nominal test item loading rate.

Executive summary:

The aim of this study was to determine the acute toxicity of the test item “OAK MOSS CONCRETE” on the daphnids Daphnia magna, according to the OECD guideline202. The daphnidswere exposed toWater Accommodated Fractions (WAFs) of the test item, following the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No.23).

 

Following the results of a preliminary study, a limit test at a nominal loading rate of 100 mg test item/L was performed, in order to demonstrate that the test item has no significant effect on the test organism at this test loading rate or that EL₅₀ at 24h and 48h is higher than 100 mg/L. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours.

 

In order to check the stability of the WAFs in the test solutions, concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). These non-specific chemical analyses were performed by Carbon Analyzer. An overview of the results is given in the table below:

Nominal test item loading rate	Renewal	Results of the determination of TOC analysis (mg/L)
		At initial time (Fresh solutions)	After 24 hours of exposure(Old solutions)	Variation(%)
WAF 100 mg/L	T0h to T24h	(1.50)* < 2	(1.71)* < 2	NA
	T24h to T48h	2.68	2.76	+ 3.0

 

*(for information)

NA: Not Applicable

 

Therefore, according to the results of the TOC analysis and since the study was carried out using WAFs of an UVCB substance, the results were based on the nominal test loading rates.

 

Validity criteria conformity:

	Values for OECD 202	Validity criteria for the test
Immobilised daphnids in control	<10%	Ok (0%)
Dissolved Oxygen concentration	>3 mg/L	Ok (8.9 mg/L)

 

 

Daphnids immobilisation

Nominal test item loading rate  in mg/L	Number of daphnids exposed at the start	Immobilised daphnids (%)
		at 24h	at 48h
WAF Control	20	0 (0%)	0 (0%)
WAF 100.0	20	0 (0%)	0 (0%)

 

Conclusion

All the validity criteria of the study were successful. The results are as follows:

- EL50-24h is higher than 100 mg/L

- EL50-48h is higher than 100 mg/L

These values are based on nominal test item “OAK MOSS CONCRETE” loading rate.

Under test conditions, no ecotoxicity was observed after 24 hours and 48 hours of exposure during the testing at 100 mg/L of the test item (loading).

 

Therefore, the results obtained for the test item “OAK MOSS CONCRETE” on the toxicological endpoint “immobilisation” give an EL₅₀-24h and an EL₅₀-48h higher than the highest tested loading rate, i.e. 100 mg/L, based on nominal test item loading rate.

The result of this study performed on an analogue is used for the registered substance.

Description of key information

Based on a read-across from an experimental GLP study performed on an analogue substance according to the OECD 202 guideline, the following result has been extrapolated to the registered substance:

-       48h-EL50 > 100 mg/L (nominal loading rate)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

For that endpoint, a study on the registered substance was not available. Therefore, in order to assess the acute toxicity of the registered substance to aquatic invertebrates, the results from an experimental OECD 202 study performed on the analogue substance Oakmoss concrete (Concrete obtained from lichen of Evernia prunastri (Parmeliaceae) by extraction with a mixture of polar and apolar solvents) have been used.

In this study, the toxic effect of the test item to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL50 (Median Effective Concentration), a statistically derived loading rate which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following the results of a preliminary study, a limit test at a nominal loading rate of 100 mg test item/L was performed, in order to demonstrate that the test item has no significant effect on the test organism at this test loading rate or that EL₅₀at 24h and 48h is higher than 100 mg/L. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours.

 

In order to check the stability of the WAFs in the test solutions, concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). These non-specific chemical analyses were performed by Carbon Analyzer. According to the results of the TOC analysis and since the study was carried out using WAFs of an UVCB substance, the results were based on nominal test loading rates.

 

Under test conditions, no ecotoxicity was observed after 24 hours and 48 hours of exposure during the testing at 100 mg/L of the test item (loading).

Therefore, the results of the study performed on the analogue substance are as follows:

- EL50-24h is higher than 100 mg/L

- EL50-48h is higher than 100 mg/L

Moreover, all the validity criteria were successful. This study complied with the guideline requirement and can therefore be considered acceptable for that endpoint.

The read-across justification is provided in the related iuclid study record.