Essential oil of Citrus sinensis (Rutaceae) peel, terpene-free

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 17 June 2020 to 4 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

xperiment conducted under GLP, following OECD guideline. No deviations were observed.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate on date of 15 November 2018 and Statement of GLP included in the sudy report

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.63, 1.12, 2.00, 3.56, 6.34 mg/L (loading rates)
- Sampling method:
Instrument: Shimadzu TOC-VWP
Calibration range: 0-5 mg.L-1
Control solutions: LOQ: 0.3 mg.L-1; QC1: 1 mg.L-1; QC2: 4 mg.L-1
Limit of quantification: LOQ: 0.3 mg.L-1
- Sample storage conditions before analysis:

Vehicle:

no

Details on test solutions:

Preparation of Water Accommodated Fractions:
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring. 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 5 L. A magnetic stirring bar was placed in each test vessel and 5.0 L to 5.5 L of test water (depending on the brim capacity of the bottles) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels 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 ca. 22 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL of solution were discarded via the drain port. Then the WAFs were directly added into test vessels containing a fixed amount of inoculum (5.103 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum headspace. At the start of the test, the test solutions in all test vessels were observed to be clear and colourless. The Tyndall effect (checked via laser beam) was negative in all treatments. The test was carried out without adjustment of the pH.


Range-finding test:
A range-finding test was conducted between June 8 and June 12, 2020 to determine the range of concentrations for the definitive test. The raw data of this test are archived under the project number of the present study.
This range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1.0, 3.2, 10.0, 32.0 and 100.0 mg.L-1 and to a control. The WAFs were in the dark under closed conditions and by slow-stirring.
The mixing vessels were graduated cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each mixing vessel and 5000 mL to 5500 mL of test water* (depending on the brim capacity of the bottles) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides (except for the loading rates of 32 and 100 mg.L-1 where test item was weighed on a weighing boat that was then placed above the mixing vessel and rinsed with test water) that were afterwards placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels 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 23 hours of gentle stirring in the dark and at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were directly added into test vessels containing a fixed amount of inoculum (5.103 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, test solutions in test vessels were observed to be clear and colourless at all loading rates. The Tyndall effect (checked via laser beam) was negative in all treatments. The test was carried out without adjustment of the pH.


Final test:Test concentrations:
Test item: Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 1.78): 0.63, 1.12, 2.00, 3.56 and 6.34 mg.L-1.
Controls: Test water without test substance but treated in the same way as the test substance solutions.
Replicates: 6 controls and 3 replicates of each loading rate for counting. Moreover, additional (abiotic) replicates of each treatment without algae were prepared for chemical analyses.

Test organisms (species):

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

Details on test organisms:

Origin: 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.

Reason for selection: This system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Stock culture: 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.

Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 10000 cells.mL-1. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Remarks:

Original medium from OECD TG 20

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

no data

Hardness:

no daa

Test temperature:

Controlled environment cabinet (23°C ± 2°C); vessels were distributed randomly in the incubator and redistributed over the test at t=24h and t=48h.

pH:

The pH of this solution was approximately in the range of 8.1 ± 0.2.

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of approximately 1.78): 0.63, 1.12, 2.00, 3.56 and 6.34 mg.L-1.

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 5.10^3 cells.mL-1
- Control end cells density: 69.4.10^4 cells.mL-1
- No. of organisms per vessel: 5.10^3 cells.mL-1
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: Continuous illumination
- Light intensity and quality: light intensity of 4,440-8,880 lux and did not vary more than ± 15% from the average light intensity over the incubation area

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Range finding study:1, 3.2, 10, 32, 100 mg/L
- Test concentrations: 0.63, 1.12, 2.00, 3.56, 6.34 mg/L
- Results used to determine the conditions for the definitive study: No effect observed at these concentrations

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

ca. 1.673 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.805 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

ca. 0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Based on the results of the range-finding test, the effect of the test item on Pseudokirchneriella subcapitata was tested in a definitive study using five loading rates.
The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based upon nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 1.673 mg.L-1 and 0.944 mg.L-1, respectively. The 72-hour EL10 for growth rate was 0.805 mg.L-1 and for yield was 0.639 mg.L-1. The 72-hour NOELR value for both parameters was 0.630 mg.L-1.

Validity criteria of the study
Cell density in controls: 138.8-fold increase within 72 hours.
Coefficient of variation:
1.The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 33.1% in 72 hours.
2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 3.0%.

Thus the validity criteria were respected in this study.

Results with reference substance (positive control):

72h-EC50 (growth rate)= 0.928 mg.L-1

Analytical results:

Concentration of dissolved organic material in thecontrols and the WAFs was checked by TOC analysis at the start and at the end of the test.TOC analyses showed that WAFs concentrations were stable between the start and the end of the test, within the ± 20% of the initial TOC concentrations values, except at the test loading rates of 0.63 mg.L-1and 1.12 mg.L-1 where 39.4% and 34.4% of TOC losses were observed,respectively.It should be recalled that the study was carried out using WAFs of a natural complex substance made of several constituents with different stabilities, solubilities and behaviours in aqueous solutions during testing. Therefore, and since the test item was a UVCB substance, the results were based on nominal loading rates.

Table 1: Mean algal cell densities in the definitive study (expressed as density of algal cells.mL-1x10^4)

	Control	0.63 mg/L	1.12 mg/L	2.00 mg/L	3.56 mg/L	6.34 mg/L
t=24h	3.5	2.0	2.7	1.5	0.3	0.2
t=48h	22.9	13.5	5.9	1.5	0.8	0.3
t=72h	69.4	63.3	20.0	3.3	0.8	0.4

Table 2: Mean specific growth rate during the final test

		Control	0.63 mg/L	1.12 mg/L	2.00 mg/L	3.56 mg/L	6.34 mg/L
t=0h-t=24h	Mean	1.896	1.238	1.643	0.971	-0.916	-1.147
t=0h-t=24h	% Inhibition	-	34.7	13.3	48.8	148.3	160.5
t=0h-t=48h	Mean	1.912	1.633	1.221	0.503	0.187	-0.343
t=0h-t=48h	% Inhibition	-	14.6	36.1	73.7	90.2	117.9
t=0h-t=72h	Mean	1.641	1.613	1.225	0.625	0.125	-0.151
t=0h-t=72h	% Inhibition	-	1.7	25.4	61.9	92.4	109.2

Table 3: Measured concentrations

Nominal concentrations (mg test item/L)	Start (t=0h)	End (t=72h)	Relative loss to initial value (t=0h-t=72h) (%)
Control	0.52	<0.3	N.A.
0.63	0.66	0.40	39.4
1.12	0.90	0.59	34.4
2.00	1.04	0.83	20.2
3.56	1.24	1.28	-3.23
6.34	2.11	2.26	-7.11

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based upon nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 1.673 mg.L-1 and 0.944 mg.L-1, respectively. The 72-hour EL10 for growth rate was 0.805 mg.L-1 and for yield was 0.639 mg.L-1. The 72-hour NOELR value for both parameters was 0.630 mg.L-1.

Executive summary:

This study was performed to assess the test item O15231 ORANGE ESS TYPE BRESIL DETERP 100% for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No.23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 0.63, 1.12, 2.00, 3.56 and 6.34 mg.L-1 and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the controls and the WAFs was checked by analysis of Total Organic Carbon (TOC) at the start and the end of the test.

TOC analyses indicate that organic compounds in the WAFs were overall stable between the start and the end of the test.

Under the experimental conditions and based upon nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 1.673 mg.L-1 and 0.944 mg.L-1, respectively. The 72-hour EL10 for growth rate was 0.805 mg.L-1 and for yield was 0.639 mg.L-1. The 72-hour NOELR value for both parameters was 0.630 mg.L-1.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

ELr50(72h)=1.673 mg/L (loading rate)

ELr10(72h)=0.805 mg/L (loading rate)

Key value for chemical safety assessment

EC50 for freshwater algae:

1.673 mg/L

EC10 or NOEC for freshwater algae:

0.805 mg/L

Additional information

One key study is available to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD guidelines 201.

This study was performed to assess the test item O15231 ORANGE ESS TYPE BRESIL DETERP 100% for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No.23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 0.63, 1.12, 2.00, 3.56 and 6.34 mg.L-1 and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the controls and the WAFs was checked by analysis of Total Organic Carbon (TOC) at the start and the end of the test.

TOC analyses indicate that organic compounds in the WAFs were overall stable between the start and the end of the test.

Under the experimental conditions and based upon nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 1.673 mg.L-1 and 0.944 mg.L-1, respectively. The 72-hour EL10 for growth rate was 0.805 mg.L-1 and for yield was 0.639 mg.L-1. The 72-hour NOELR value for both parameters was 0.630 mg.L-1.