Concrete of honeycomb cells of the bee by hexane extraction

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:

from 18 September to 27 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted March 23, 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 4, 5 and 6 July 2016 / Certificate signed on January 10th, 2017

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry area, unopened containers, optimum temp. 11-25°C

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Duplicate samples for analysis were taken from the control and the limit test loading rate (from a replicate of each treatment without algae dedicated exclusively to chemical analyses) at the start and the end of the test. Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs. TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Vehicle:

no

Remarks:

In the final test, heating and sonication were used to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible.

Details on test solutions:

Preparation of test solution (final test) :
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring. After having tried different methods for the preparation of the WAFs in the preliminary study (cf. Appendix V), the protocol described hereafter has been selected for the final test.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 1 L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace). The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. Each glass flask was placed in a water bath for 10-15 minutes at approx. 50°C, followed by sonication for approx. 10 minutes. Based on experience on similar substances, the heating/sonication step is a method allowing to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible.
Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 22 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test flasks containing a fixed amount of inoculum (5.103 cells/mL per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, the solution was observed to be clear and colourless.

Preparation of test solutions (range-finding test) :
The range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1, 10, 32 and 100 mg/L and to a control, according two methods of preparation ((a) use of solvent (acetone); (b) heating/sonication). The WAFs were prepared in the dark under closed conditions and by slow-stirring.
The mixing vessels were 1 L cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace).
(a) The loading rates of the test item were weighed in glass vials and resuspended in acetone. Then the loading rates of the test item/vehicle were vigorously vortexed for at least 5 min*, and thereafter a specific volume (corresponding to a (limit) solvent concentration of 100 mg/L, as recommended in OECD guidance on testing and assessment No. 23) of each preparation was carefully added to the surface of test water contained in the mixing vessels that were closed immediately.
*Despite this, it was not possible to dissolve satisfactory all the beeswax in acetone, especially for the loading rate of 100 mg/L (the preparation was not liquid, and thus not harvestable), where finally an acetone concentration of 1000 mg/L was used (instead of 100 mg solvent/L).
(b) The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. Each glass flask was placed in a water bath for 10 minutes at approx. 50°C, followed by sonication for 10 minutes. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately.

In both cases (a) and (b), the mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test flasks containing a fixed amount of inoculum (5.103 cells/mL per vessel) that were immediately sealed after filling with a minimum of headspace. The test was carried out without adjustment of the pH.
Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs (only for (b)).


PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: Test water without test substance but treated in the same way as the test substance solution.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): In method (a), the maxiumum recommended solvent concentration was carefully added to the surface of test water

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata, CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Age of inoculum (at test initiation):
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.

Test type:

static

Water media type:

other: sterilised water

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

The temperature in the incubator was situated between 23.4 and 23.9°C throughout the test (average value: 23.8°C).

pH:

Between 8.19 and 9.90

Nominal and measured concentrations:

100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: cylindrical glass bottles sealed with screw caps
- Type (delete if not applicable): closed
- Headspace, fill volume: minimum headspace, 1 L fill volume

- Initial cells density: 5.103 cells/mL
- No. of vessels per concentration (replicates): 6 replicates (limit test)
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per vehicle control (replicates): 1 vessel in the range-finding test

GROWTH MEDIUM
- Standard medium used: yes, Original medium of OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: No
- Photoperiod: continuous illumination
- Light intensity and quality: 4 440-8 880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber (at t=0h, t=24h and t=48h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study : the concentrations tested were 1.0, 10.0, 32.0, 100.0 mg/L.
- Test concentrations: A limit test was performed at 100 mg/L (loading).
- Results used to determine the conditions for the definitive study: Since no significant effect was observed at any of the tested loading rates in the range-finding test, it was decided to perform a limit test at 100 mg/L (loading) for the final study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

The 72h-EC50 was 1.457 mg/L for the parameter growth rate.

Reported statistics and error estimates:

No statistical analysis was performed. Effective concentrations were determined directly from the raw data.

Table 1: Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L^-1) - Final (limit) test.

 

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	0.88	0.47
Control	0.92	0.43
100	1.12	0.82
100	1.07	0.66

* WAF prepared at the given loading rate.

Table 2: Algal cell densities during the final test (expressed as density of algal cells.mL^-1x10⁴).

	Replicate	Nominal concentration (mg test item.L^-1)*
		Control	100
t=24h	1	4.4	4.0
	2	4.0	4.8
	3	7.2	6.0
	4	4.4	4.4
	5	4.8	4.4
	6	4.0	4.0
	Mean	4.8	4.6
	Std. Dev.	1.21	0.75
	% Inhib.	-	1.4
t=48h	1	22.0	25.6
	2	21.2	24.0
	3	26.0	22.0
	4	18.0	23.2
	5	22.0	24.4
	6	20.0	24.0
	Mean	21.5	23.9
	Std. Dev.	2.66	1.20
	% Inhib.	-	-2.9
t=72h	1	86.0	96.8
	2	91.6	92.0
	3	84.4	92.8
	4	74.0	94.0
	5	93.6	86.8
	6	90.0	89.6
	Mean	86.6	92.0
	Std. Dev.	7.06	3.48
	% Inhib.	-	-1.2

* WAF prepared at the given loading rate.

% Inhib.: %Inhibition of growth rate relative to the control determinedby the computer program ToxRat (8).

At test start 5000algal cells.mL^-1were incubated; 6 replicates of the controls and 6 replicates of the limit test loading rate.

Std. Dev.: standard deviation.

Table 3: pH values during the final test

	Nominal concentration (mg test item.L^-1)*
	Control	100
Start t=0h	8.23	8.19
End t=72h	9.75	9.90

* WAF prepared at the given loading rate.

Validity criteria fulfilled:

yes

Remarks:

There was a minor deviation : the pH level in the control varied more than 1.5 units at the end of the test (1.52 units of difference) this was not considered to have affected the integrity of the study.

Conclusions:

Based on nominal concentrations, the 72-hour EC50 and 72-hour EC10 values for the parameters growth rate and yield were considered to be higher than 100 mg/L.

Executive summary:

The influence of the registered substance on algal growth inhibition (P. subcapitata) was investigated according to OECD guideline 201. Algae were exposed to the test substance under closed and static conditions during 72 hours.

Based on the results of a range-finding test, a limit test was performed at a concentration of 100 mg/L (nominal). 6 replicates were performed for the limit test at the loading rate of 100 mg/L and for the control. Concentration of dissolved organic material in the control and the limit test loading rate was checked by TOC analysis at the start and the end of the test.

Based on nominal concentrations, the 72-hour ErC₅₀ and 72-hour ErC₁₀ values for the parameter growth rate and yield were estimated to be higher than 100 mg.L^-1.

Description of key information

OECD 201, GLP, key study, validity1:

72h-ErC50 (Pseudokirchneriella subcapitata) > 100 mg/L based on nominal concentration

72h-ErC10 (Pseudokirchneriella subcapitata) > 100 mg/L based on nominal concentration

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

One key study is available to assess the toxicity of the registered substance to aquatic algae Pseudokirchneriella subcapitata. This study was performed according to the OECD guideline 201, with GLP compliance under closed and static conditions for 72 hours.

Based on the results of a range-finding test, a limit test was performed at a concentration of 100 mg/L (nominal). 6 replicates were performed for the limit test at the loading rate of 100 mg/L and for the control.Concentration of dissolved organic material in the control and the limit test loading rate was checked by TOC analysisat the startand the end of the test.

Based on nominal concentrations,the 72-hour ErC₅₀and 72-hour ErC₁₀values for the parameter growth rate and yield were estimated to be higher than 100 mg.L^-1.