Essential oil Schinus Terebinthifolius (Anacardiaceae) obtained from red berries by supercritical carbon dioxide extraction (indicative)

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:

10-14 November 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This GLP study was performed according to OECD Guideline 202 with the following deficiencies: old WAF method - dispersions were stirred for 3 hours and then filtered ; the 48-hour EL50 was determined as the geometric mean value of the two consecutive test concentrations with 0 and 100% immobility (EL0 and EL100) as no dose/response curve were observed after 48 hours.

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)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

The Swiss GLP Monitoring Authorities (inspected on 05-09 & 26-30 November 2007 / signed on 12 November 2008)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance: Pale yellow liquid

Analytical monitoring:

yes

Details on sampling:

Analysis of the test Item concentrations
For the characterization of the WAFs, duplicate samples were taken from each treatment before the test start and at the end of the test after 48 hours.
For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling.
Immediately after sampling, acetone was added to each sample (1% of acetone per sample volume). All samples as listed below were analyzed immediately after sampling.
The concentrations of the test item, based on the sum of four peaks, were analyzed in the duplicate test media samples of all loading rates from both sampling times (0 and 48 hours).
From the control, only one of the duplicate samples was analyzed from each of the sampling times.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared (table 6.1.3/1)
The dispersions were stirred for 3 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters (Schleicher & Schuell, Type NC45, pore size 0.45 μm) and the undiluted filtrates were tested as WAFs. The negative pressure of the filtration unit was reduced as much as possible to avoid losses of volatile components of the test item during filtration.
The stirring period of 3 hours was chosen based on the results of pre-experiments (non-GLP) in which the maximum concentration of the different compounds in the test media was reached after the stirring period of 3 hours.
The test media were prepared just before the start of the test.
- Controls: Test water without test item

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna Straus clone 5
- Source: A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Age: At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.
- Feeding during test: Daphnids were not fed during the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

Water Hardness: 250 mg/L as CaCO3

Test temperature:

20 °C

pH:

At the beginning and end of the test period, pH value of the test media was in the range of 7.5 to 7.8

Dissolved oxygen:

At the beginning and end of the test period, the dissolved oxygen concentrations in the test media and control were at least 7.1 mg/L.

Salinity:

No data

Nominal and measured concentrations:

Water accommodated fractions (WAFs) with the loading rates of 4.6, 10, 22, 46 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50-mL glass tubes
- Type: closed; the test tubes were made tight with glass stoppers to avoid losses of the volatile substance.
- Material, size, headspace, fill volume: Since the test item was determined to be volatile (according to the results of a pre-experiment, non-GLP), the test was performed in 50-mL glass tubes completely filled with about 50 mL of test medium.
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- A static test without test medium renewal was performed.
- The volume of test solution provided for each daphnia was 10 mL. Thus, the requirement of the test guidelines for the minimum volume of 2 mL test medium per daphnia was fulfilled. The daphnids were randomly distributed to the test vessels at initiation of the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO 6341 was used in the study.
- Alkalinity: 0.8 mmol/L
- Ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity

OTHER TEST CONDITIONS
- Test was performed as far as possible in the dark to avoid a photolytic degradation of the test item.

EFFECT PARAMETERS MEASURED
- Determination of the Immobility: The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Those daphnids that were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized.
- Water Quality Criteria: At the start and end of the test, the pH values, dissolved oxygen concentrations and water temperature were determined at each test concentration and in the control. The appearance of the test media was visually recorded at the start of the test and after 24 and 48 hours.

RANGE-FINDING STUDY
- The selection of the loading rates was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item in the test water (non-GLP).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 10 and 22 mg/L

Remarks:

geometric mean value of the two consecutive test concentrations with 0 and 100% immobility (EL0 and EL100)

Duration:

48 h

Dose descriptor:

other: The highest tested loading rate without observed effect

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the loading rate of 10 mg/L. From the loading rate of 22 mg/L onwards, all daphnids were found to be immobile. Thus, the concentration-effect relationship was very steep. The 24-hour EL50 of the test item was calculated to be 15 mg/L with 95% confidence limits of 10 and 22 mg/L. The 24-hour EL0 was 10 mg/L. The 24-hour EL100 was 22 mg/L.
- Additionally to the immobilization recorded after 24 hours, one daphnid was found to be immobile at the loading rate of 10 mg/L (5% immobility) after 48 hours of exposure. This low immobilization rate was estimated as a significant toxic effect, but had no influence on the calculation of the 48-hour EL50. The 48-hour EL50 was calculated to be 15 mg/L with 95% confidence limits of 10 and 22 mg/L. The 48-hour EL0 and the 48-hour NOEC (highest concentration tested without toxic effects after 48 hours) was 4.6 mg/L, since no significant immobilization was observed up to and including this test concentration. The 48-hour EL100 was 22 mg/L.
- No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.

Results with reference substance (positive control):

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in March 2008 (48-hour EC50: 0.73 mg/L, RCC Study No. B83744) indicated that the sensitivity of the test organisms was within the historical range of Harlan Laboratories (48-hour EC50 from 1996 to 2008: 0.53-1.1 mg/L).

Reported statistics and error estimates:

The 24- and 48-hour EL50 could not be calculated by Probit Analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the 48-hour EL50 was determined as the geometric mean value of the two consecutive test concentrations with 0 and 100% immobility (EL0 and EL100), and the confidence limits for the EL50 as the test concentrations with 0 and 100% immobility.
The NOEC, EL0 and EL100 were determined directly from the raw data.

Table 6.1.3/2: Effect of test item on the Mobility of Daphnia magna

Loading Rate (mg/L)	No. of daphnids tested	Immobilized daphnids after 24 hours		Immobilized daphnids after 48 hours
		No.	%	No.	%
Control	20	0	0	0	0
4.6	20	0	0	0	0
10	20	0	0	1	5
22	20	20	100	20	100
46	20	20	100	20	100
100	20	20	100	20	100

 

At the start of the test, the measured test item concentrations (based on the sum of four peaks) in the test media with the loading rates of 4.6, 10, 22, 46 and 100 mg/L were 0.30, 0.80, 2.1, 3.2 and 4.2 mg/L, at the end of the test 0.17, 0.49, 1.1, 1.3 and 2.2 mg/L, respectively.

Since water accommodated fractions of the test item were tested, all reported biological results were based on the loading rates of the test item.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 48-hour EL50 of the test item was calculated to be 15 mg/L (95% confidence limits of 10 and 22 mg/L).

Executive summary:

A study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the test item to Daphnia magna, under static conditions.

 

In order to assess the toxicity of test item containing different components to daphnids, water accommodated fractions (WAFs) with the loading rates of 4.6, 10, 22, 46 and 100 mg/L were tested. Additionally, a control (test water without test item) was tested in parallel.

For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 3 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters and the undiluted filtrates were tested as WAFs. The negative pressure of the filtration unit was reduced as much as possible to avoid losses of volatile components of the test item during filtration. The stirring period of 3 hours was chosen based on the results of pre-experiments (non-GLP) in which the maximum concentration of the different compounds in the test media was reached after the stirring period of 3 hours.

 

At the start of the test, the measured test item concentrations (based on the sum of four peaks) in the test media with the loading rates of 4.6, 10, 22, 46 and 100 mg/L were 0.30, 0.80, 2.1, 3.2 and 4.2 mg/L, at the end of the test 0.17, 0.49, 1.1, 1.3 and 2.2 mg/L, respectively. Since water accommodated fractions of the test item were tested, all reported biological results were based on the loading rates of the test item.

 

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the loading rate of 10 mg/L. From the loading rate of 22 mg/L onwards, all daphnids were found to be immobile. Thus, the concentration-effect relationship was very steep. Additionally to the immobilization recorded after 24 hours, one daphnid was found to be immobile at the loading rate of 10 mg/L (5% immobility) after 48 hours of exposure. This low immobilization rate was estimated as a significant toxic effect, but had no influence on the calculation of the 48-hour EL50. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.

 

Under the test conditions, the 48-hour EL50 of the test item was calculated to be 15 mg/L (95% confidence limits of 10 and 22 mg/L).

Description of key information

OECD Guideline 202, GLP, key study, validity 2:

WAF, 48h-EL50 (Daphnia magna) = 15 mg/L (95% CL: 10 - 22 mg/L), based on nominal loading rates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

15 mg/L

Additional information

To assess the short-term toxicity of the registered substance to aquatic invertebrates, one key study is available.

This study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the registered substance (Item 2) to Daphnia magna, under static conditions. Water Accommodated Fractions (WAFs) with the loading rates of 4.6, 10, 22, 46 and 100 mg/L were tested as the registered substance is a UVCB substance. A control (test water without test substance) was tested in parallel. At the start of the test, the measured test substance concentrations (based on the sum of four peaks) in the test media with the loading rates of 4.6, 10, 22, 46 and 100 mg/L were 0.30, 0.80, 2.1, 3.2 and 4.2 mg/L, at the end of the test 0.17, 0.49, 1.1, 1.3 and 2.2 mg/L, respectively. Since WAF of the test substance were tested, all reported biological results were based on the loading rates of the test substance. 

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the loading rate of 10 mg/L. From the loading rate of 22 mg/L onwards, all daphnids were found to be immobile. Thus, the concentration-effect relationship was very steep. Additionally to the immobilization recorded after 24 hours, one daphnid was found to be immobile at the loading rate of 10 mg/L (5% immobility) after 48 hours of exposure. This low immobilization rate was estimated as a significant toxic effect, but had no influence on the calculation of the 48-hour EL50. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration. 

Under the test conditions, the 48-hour EL50 of the test substance was calculated to be 15 mg/L (95% confidence limits of 10 and 22 mg/L), corresponding to the geometric mean value of the two consecutive loading rates with 0 and 100% immobility.