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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 June 2020 to 6 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate dated on November 15 2018 and GLP statement included in study report

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from the control and all test solutions at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs.

Vehicle:

no

Details on test solutions:

Nominal loading rates (spaced by a factor of 1.7): 2.0, 3.4, 5.8, 9.8, 16.7 and 28.4 mg.L-1

Preparation of stock Water Accommodated Fractions
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for freshmedia at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production
of a dispersion.
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 (except for the loading rates of 16.7 and 28.4 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 24 ± 2 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 were discarded via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The 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 (for fresh media at t=0h and t=24h). The test was carried out without adjustment of the pH.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: female, clone 5
- Age at study initiation (mean and range, SD): < 24 hours old
- Method of breeding: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1, and once week with an additional suspension of Spirulina. The water was changed at least once a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes
- Age of parental stock (mean and range, SD):
- Feeding during test : no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

no data

Test temperature:

the temperature of the test medium was situated between 20.6 and 21.2°C throughout the test (average value: 21.0°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

pH:

7.85-8.66

Dissolved oxygen:

7.65-8.96 mgO2/L

Salinity:

not applicable

Nominal and measured concentrations:

nominal loading rates (spaced by a factor of 1.7): 2.0, 3.4, 5.8,9.8, 16.7 and 28.4 mg.L-1

Details on test conditions:

TEST SYSTEM
- Test vessel: flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Type: closed
- Volume of solution: 60 ml
- Aeration: No aeration of the test solutions occurred throughout the test.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: 16h light : 8h dark
- Light intensity: no data


RANGE-FINDING STUDY
- Test concentrations: 0.32, 1.0, 3.2, 10.0, 32.0 and 100.0 mg.L-1

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

10.819 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

12.672 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure, immobilisation rates were 0% at 2.0, 3.4 and 5.8 mg.L-1 (loading rates), 15% at 9.8 mg.L-1 and 100% at 16.7 and 28.4 mg.L-1(loading rates). Therefore, the highest loading rate without effect and the 48h no observed effect concentration (NOELR) was 5.8 mg.L-1.
The EL50 values using probit analysis were determined by the computer program ToxRat (8), and were as follows at each of the observation times:
EL50(24h)=12.672 mg/L (95%CL: 11.695-13.759 mg/L) (loading rates)
EL50(48h)=10.819 mg/L (95%CL: n.d.) (loading rates)

Validity criteria of the study
Controls: In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.
Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 3 mg.L-1 incontrols and test vessels.
Thus the validity criteria have been fulfilled in the present study.

Results with reference substance (positive control):

24h-EC50 = 1.365 mg.L-1.

Water quality parameters and environmental conditions throughout the test

The results of measurement of pH and oxygen concentrations (mg.L-1) remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: ≥ 3 mg.L-1 at the end of the test).

Furthermore, the temperature of the test medium was situated between 20.6 and 21.2°C throughout the test (average value: 21.0°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

Analytical results

The results of analysis of the samples taken during the final test are described in the Table below. Concentration of dissolved organic material in the controls 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).

TOC analyses showed that organic compounds in the loading rates were found to be relatively stable between the start and the end of first exposure period (t=0h-t24hOld), and a little less stable during the second part of the test (t=24hFresh-t=48h) (see Table below). However, it was considered that test item concentrations were maintained as much as possible since test solutions were renewed at t=24h. In any case, due to the complex nature of the WAF and since the test item was a UVCB substance, the results were based on nominal loading rates and as Effective Loading Rate 50, according to the OECD No. 23 (7).

Nominal concentration (mg test item /L)	Start (t=0h)	t=24h Old	Relative loss to initial value (t=0h, t=24hold) (%)	t=24h Fresh	End (t=48h)	Relative loss to initial value (t=24hfesh-t=48h) (%)
Control	1.17	1.56	-33	0.96	0.82	15
2.0	1.64	1.57	4	1.75	1.23	30
3.4	2.01	2.42	-20	2.18	1.68	23
5.8	2.31	2.88	-25	3.34	2.19	34
9.8	3.66	3.37	8	5.07	3.24	36
16.4	6.65	7.53	-13	na	na	na
28.4	9.21	10.30	-12	na

na: no applicable because no further analysis was performed for concentration in which all daphnids were immobilised

Biological results

Acute immobilisation of daphnids after 24 and 48 hours in the final test

Nominal concentrations (mg test item/L)	Immobilisation at 24h %	Immobilisation at 48h %
Control	0	0
2.0	0	0
3.4	0	0
5.8	0	0
9.8	0	15
16.7	100	100
28.4	100	100

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of test item to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under experimental conditions, and based upon nominal loading rates, the 48-hour EL50 value was estimated to be 10.819 mg.L-1.

Executive summary:

A study was performed to assess the acute toxicity of test item O15231 ORANGE ESS TYPE BRESIL DETERP 100% to Daphnia magna. 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 aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the 48 h EL50 (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 2.0, 3.4, 5.8, 9.8, 16.7 and 28.4 mg.L-1, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the control and the WAFs was checked by analysis of Total Organic Carbon (TOC) at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

TOC analyses indicate that organic compounds in the WAFs were stable the first day of the test, and slightly less stable the second day (losses of approx. 23-36%). However, it was considered that test item concentrations were maintained as much as possible since test solutions were renewed at t=24h. After 48 hours of exposure, immobilisation rates were 0% at 2.0, 3.4 and 5.8 mg.L-1, 15% at 9.8 mg.L-1 and 100% at 16.7 and 28.4 mg.L-1.

The toxic effect of O15231 ORANGE ESS TYPE BRESIL DETERP 100%to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under experimental conditions, and based upon nominal loading rates, the 48-hour EL50 value was estimated to be 10.819 mg.L-1.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

EL50(48h)=10.819 mg/L (loading rate)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10.819 mg/L

Additional information

One key study is available to assess the effect of the test material on the mobility of the aqutic invertebrate Daphnia magna. The method followed that described in the OECD guidelines 202.

This study was performed to assess the acute toxicity of test item O15231 ORANGE ESS TYPE BRESIL DETERP 100% to Daphnia magna. 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 (2) and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the 48 h EL50 (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 2.0, 3.4, 5.8, 9.8, 16.7 and 28.4 mg.L-1, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the control and the WAFs was checked by analysis of Total Organic Carbon (TOC) at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

TOC analyses indicate that organic compounds in the WAFs were stable the first day of the test, and slightly less stable the second day (losses of approx. 23-36%). However, it was considered that test item concentrations were maintained as much as possible since test solutions were renewed at t=24h. After 48 hours of exposure, immobilisation rates were 0% at 2.0, 3.4 and 5.8 mg.L-1, 15% at 9.8 mg.L-1 and 100% at 16.7 and 28.4 mg.L-1.

The toxic effect of O15231 ORANGE ESS TYPE BRESIL DETERP 100%to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under experimental conditions, and based upon nominal loading rates, the 48-hour EL50 value was estimated to be 10.819 mg.L-1.