Concrete obtained from lichen of Pseudevernia furfuracea (syn evernia furfuracea)(Parmeliaceae) by extraction with a mixture of polar and apolar solvents

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-03 to 2017-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to the OECD 201 guideline. All validity criteria were successful.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 2017-11-22

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

In order to verify maintenance of the WAFs during the ecotoxicological testing, chemical analyses of test item were performed in the test solutions (biotic and abiotic replicates):
- at the beginning of the test (T = 0h)
- at the end of the test (T = 72h)

Schedule of analysis at initial time T0h:
- WAF Control Abiotic
- WAF 100.0 mg/L Abiotic

Schedule analysis at final time T72h:
- WAF Control Biotic
- WAF 100.0 mg/L Biotic
- WAF Control Abiotic
- WAF 100.0 mg/L Abiotic

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.
Once 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 at 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: the solution obtained was named WAF (Water Accommodated Fraction).

TEST SOLUTIONS
Two non-GLP preliminary studies (with and without solvent) have been performed in order to select the method for the test solution preparation and the concentrations to be used in the final test. Based on these results, the final test was performed at the limit test item concentration of 100 mg/L, and without solvent.
For the non-GLP preliminary test with solvent, DMSO has been determined as the most suitable solvent to be used. For stock solution at 100 g test item /L DMSO, 4 loading rates 1; 10; 50 and 100 mg/L were prepared before test with algae. 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 could not be selected for the final test.

The final test contained:
- Control: 6 biotic replicates + 2 abiotic replicates
- Concentration 100 mg/L: 6 biotic replicates + 2 abiotic replicates

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: chlorococcal order strain number CCAP 278.4
The algae cultures are stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).

Test type:

static

Water media type:

freshwater

Remarks:

The medium was prepared from concentrated stock solutions in ultrapure water as described in the guideline OECD 201.

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

22.8 °C – 23.3°C

pH:

8.0 - 8.1

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Limit test: 100 mg test item /L (nominal, loading rate)

Details on test conditions:

TEST SYSTEM
In agreement with the study monitor and according to the results from the preliminary tests, this study was conducted with one concentration level: 100.0 mg/L: Limit test, WAF conditions.
A growing exponential phase inoculum of the freshwater unicellular alga Pseudokirchneriella subcapitata was added in order to obtain an initial concentration of 10^4 cells/mL at the start of the test: 100 mL of algal cultures grown in 250 mL erlenmeyer flasks which were placed in a cooling incubator at a temperature in the range 21 to 24°C controlled at ± 2°C and under a constant shaking fixed at 120 rpm and a continuous illumination comprised between 4 440 and 8 880 lux.
The algal growth inhibition in relation to the control was evaluated over a period of 72 hours. The algal cells per unit volume were measured during the test at 24, 48 and 72 hours with a particle counter (Beckman Coulter Z2) and/ or under microscope with Malassez counting cell.
The pH of each test concentration was measured at the start and at the end of the test. The pH of the control medium had not increased by more than 1.5 units during the test.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS.
The medium was prepared from concentrated stock solutions in ultrapure water as described in the guideline
Algal dilution water
Theoretical composition
NH4Cl 15 mg/L
MgCl2, 6H20 12 mg/L
CaCl2, 2H2O 18 mg/L
MgSO4,7H20 15 mg/L
KH2PO4 1.6 mg/L
FeCl3, 6H2O 0.08 mg/L
Na2EDTA, 2H2O 0.1 mg/L
H3BO3 0.185 mg/L
MnCl2, 4H2O 0.415 mg/L
ZnCl2 3 µg/L
CoCl2, 6 H20 1.5 µg/L
CuCl2, 2H2O 10-2 µg/L
Na2MoO4, 2H2O 7 µg/L
NaHCO3 50 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Light intensity and quality: 6461 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beckman Coulter Z2 and Malassez counting cell

TEST CONCENTRATIONS
- 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:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Results with reference substance (positive control):

The last algal growth inhibition test performed with the reference item K2Cr2O7 (September 18 2017) gave an ErC50(0-72h) = 1.11 mg/L (0.98 – 1.24), indicating that the algae sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the International Standard NF EN ISO 8692 from Mai 2012 as mentioned in the OECD Guideline) and in accordance with previous data obtained by the laboratory.

Reported statistics and error estimates:

Not applicable.

Validity criteria for the test

	Criteria for OECD 201	Validity criteria conformity
Increase biomass during 72 hours	Factor of at least 16	Yes (164.7)
Daily section-by-section specific growth rate CV	< 35%	Yes (17.2%)
Average specific growth rate CV during 72 hours	< 7%	Yes (1.0%)

 

All the validity criteria were successfully validated.

Algal growth inhibition

Raw data,i.e. algal concentrations values expressed as cell number *10⁴per mL of test solutions after 24, 48 and 72 hours of incubation are indicated in the following table.

Nominal test item loading rate		[algae] *10^4/mL at 24 hours			[algae] *10^4/mL at 48 hours			[algae] *10^4/mL at 72 hours
		Count.	Average	CV	Count.	Average	CV	Count.	Average	CV
WAF Control	a	5,1	4.5	11.7	22,3	22.1	8.1	153,8	164.7	5.0
	b	4,5			21,4			162,4
	c	4,3			21,1			169,1
	d	4,0			24,3			157,2
	e	5,2			23,8			172,8
	f	4,0			19,5			173,1
WAF 100 mg/L	a	4,4	4.0	16.5	26,5	22.8	20.7	148,7	149.3	20.7
	b	3,5			19,1			125,1
	c	4,1			30,4			134,2
	d	3,4			18,1			117,5
	e	3,4			21,5			171,3
	f	5,0			21,2			199,1

CV: Coefficient of variation (%)

The coefficients of variation for section-by-section specific growth rates in the control cultures are presented in the following table to calculate one of the validity criterion: average value of coefficient of variation (Average CV).

 

Control replicates		Specific growth rate
		0-24h	Average	CV (%)	24 -48h	Average	CV (%)	48 -72h	Average	CV (%)	0 - 72h	Average	CV per replicate (%)	Average CV
WAF Control	a	1,629	1,502	7,7	1,475	1,589	7,2	1,931	2,012	5,6	1,679	1,701	13,8	17.2%
	b	1,504			1,559			2,027			1,697		16,9
	c	1,459			1,591			2,081			1,710		19,2
	d	1,386			1,804			1,867			1,686		15,5
	e	1,649			1,521			1,982			1,717		13,9
	f	1,386			1,584			2,183			1,718		24,2

CV: Coefficient of variation (%)

 Chemical analysis of algal solutions

The table below summarizes the results achieved and the variation between T0 and T72 hours.

Nominal test item loading rate	mg “NPOC”/L in algae solutions		Variation between T0 and T72 hrs (%)
	Results at T0 hour	Results at T72 hours
WAF Control Biotic	/	5.02	NA
WAF 100 mg/L Biotic	/	4.34	NA
WAF Control Abiotic	< 2 (0.56)*	3.02	NA
WAF 100 mg/L Abiotic	< 2 (0.85)*	3.26	NA

*(for information)

 pH values and observations

Nominal test item loading rate (mg/L)	pHat the start (biotic replicates)	pHafter 72 hours (biotic replicates)
WAF Control	8.0	7.4
WAF 100	7.9	7.4

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item “TREE MOSS CONCRETE” on the toxicological endpoint “inhibition of growth rate” indicate an ErL50-(0-72h) 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 effects of the test item "TREE MOSS CONCRETE" on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011). The algae were exposed to Water 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 72h is higher than 100 mg/L (loading). The effects on the growth of the algae was determined by cell counting daily during 72 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) and at the end of the test (t=72h). These non-specific chemical analyses were performedby Carbon Analyzer.

 

The table below summarizes the achieved results and the variation between T0 and T72 hours.

 

Nominal test item loading rate	mg “NPOC”/L in algae solutions		Variation between T0 and T72 hrs (%)
	Results at T0 hour	Results at T72 hours
WAF Control Biotic	/	5.02	NA
WAF 100 mg/L Biotic	/	4.34	NA
WAF Control Abiotic	< 2 (0.56)*	3.02	NA
WAF 100 mg/L Abiotic	< 2 (0.85)*	3.26	NA

*for information

NA: not applicable

 

All measured values were lower than 2 mg/L (=LOQ) and are notified for information (in brackets). The slight increase after 72 hours of exposition was mainly due to the gaseous exchange.

 

TOC analyses indicated that organic compounds were found below the LOQ in the control and in the WAFs at 100 mg/L. Therefore, the maintenance of the WAFs throughout the test, cannot be accurately evaluated. Nevertheless, since WAFs are by definition a complex mixture and since the test item is an UVCB substance, the results are based on the nominal test loading rates.

 

The results of this study are summarized below:

- Test item: TREE MOSS CONCRETE

- Test system: Pseudokirchneriella subcapitata

- Exposure duration: 72 hours

- Test concentrations: Control - 100 mg/L

- Incubator: Temperature 22.8 – 23.3 °C ; shaking at 120 rpm; light intensity: 6461 Lux

- Measurement of cell growth: Beckman Coulter Z2 and Malassez counting cell

- Validity criteria: In the control. the increase of the biomass during 72 hours was determined to be higher than factor of 16.The coefficients of variation of daily section-by-section specific growth rateand ofaverage specific growth ratewere determined respectively to be less than 35% and 7% in the control. - The validity criteria were successfully fulfilled.

- Results: The results obtained for thetest item “TREE MOSS CONCRETE”on the toxicological endpoint “inhibition of growth rate” indicate an ErL₅₀-(0-72h) higher than the highest tested loading rate, i.e. 100 mg/L, based on nominal test item loading rate.

The results obtained for the test item “TREE MOSS CONCRETE”on the toxicological endpoint “Yield inhibition” indicate anEyL₅₀-(0-72h)higher than the highest tested loading rate, i.e. 100 mg/L, based on nominal test item loading rate.

Description of key information

The results obtained for the test item on the toxicological endpoint “inhibition of growth rate” indicate an ErL₅₀-(0-72h) higher than the highest tested loading rate, i.e. 100 mg/L, based on nominal test item loading rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

The results of this study are summarized below:

- Test item: TREE MOSS CONCRETE

- Test system: Pseudokirchneriella subcapitata

- Exposure duration: 72 hours

- Test concentrations: Control - 100 mg/L (limit test)

- Incubator: Temperature 22.8 – 23.3 °C ; shaking at 120 rpm; light intensity: 6461 Lux

- Measurement of cell growth: Beckman Coulter Z2 and Malassez counting cell

Results: The results obtained for the registered substance on the toxicological endpoint “inhibition of growth rate” indicate an ErL₅₀-(0-72h)higher than the highest tested loading rate,i.e. 100 mg/L, based on nominal test item loading rate.

The results obtained for thetest item “TREE MOSS CONCRETE” on the toxicological endpoint “Yield inhibition” indicate an EyL₅₀-(0-72h) higher than the highest tested loading rate, i.e. 100 mg/L, based on nominal test item loading rate.

Validity criteria: In the control, the increase of the biomass during 72 hours was determined to be higher than factor of 16.The coefficients of variation ofdaily section-by-section specific growth rateand ofaverage specific growth ratewere determined respectively to be less than 35% and 7% in the control. The validity criteria were successfully fulfilled.

This study respected the requirements of the test guideline. The study was considered acceptable for that endpoint.