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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29th March 2007 - 1 June 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
GLP compliance:
yes
Specific details on test material used for the study:
- Purity: 100%.
- Lot/Batch No.: 1215
- Expiration date of the lot: 30 March 2009
- Storage condition of test material: Ambient, in the dark.
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Four samples (approx. 20 ml) were taken from the freshly-prepared control and test media on each occasion (0 and 24 hours). Duplicate samples (approx. 20 ml) were also taken from the expired (24 hours old) medium in each vessel after 24 and 48 hours. Samples for chemical analysis were in vessels that were completely filled and sealed with no headspace.
- Sample storage conditions before analysis: On each occasion, the samples were stored in a refrigerator for up to twelve days until duplicate samples of fresh media and individual samples of expired media from each vessel were analysed. The remaining samples were held refrigerated in case further analysis was required.
Vehicle:
no
Details on test solutions:
As the test substance was a complex mixture comprising many components with a range of different molecular weights, the Daphnia were exposed to Water Accomodated Fractions (WAFs) of the test substance.

- Method: The method of preparation used during the definitive test was based on the results of a range finding test. On each occasion, glass bottles (total capacity, ca. 2.68 l) were completely filled with dilution medium (softened Elendt M4, which was prepared in de-ionised water) and sealed using a screw cap fitted with a silicone septum. The contents of the vessels were stirred at a rate capable of creating a vortex of approximately 5% of the static depth of the medium in each vessel before they were sealed. The test substance (15, 30 or 60 μl) was injected through the silicone septum into stirring dilution medium to give media with nominal Tea Tree Oil loading rates of 5, 10 and 20 mg/l (corrected for specific gravity, 0.89 g/l; HLS Study No. CSV/0010). The preparation vessels were covered with black bags and left to stir overnight.

On cessation of stirring, the media were left to stand in the test area for approximately four hours before 20 ml of each WAF was removed and discarded. The required volume of medium (water accomodated fraction; WAF) was then siphoned from each bottle and either used as the test medium or further diluted with dilution medium to provide the test medium at 0.625, 1.25 or 2.5 mg/l. Aliquots of the media were placed into the test vessels (ca. 130 ml glass reaction vials), which were completely filled and sealed leaving no headspace.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Strain: Straus
- Source: Cultured in-house. Obtained from a strain originating from the Institute National de Recherche Chimique Appliqué (IRChA), France.
- Age at study initiation: Less than 24 hours old.
- Method of breeding: Culture conditions ensure that the stock animals reproduce by parthenogenesis.
- Food type: A suspension of the unicellular green alga, Pseudokirchneriella subcapitata.
- Amount: Enough to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given.
- Frequency: Daily.

- Environmental conditions: Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.8 litres of softened Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2°C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week.

- Other: The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The total hardness of the dilution medium was measured before use; it was within acceptable limits.
Test temperature:
The temperature of the test area was maintained at 20.6 to 21.8°C during the definitive test, which was within acceptable limits. Temperature was continuously monitored in an additional vessel containing the same volume of dilution medium. The temperature of control and test media was measured on the fresh medium remaining in the preparation vessels and on the expired medium in each test vessel on each occasion during the study.
pH:
The pH of control and test media was measured on the fresh medium remaining in the preparation vessels and on the expired medium in each test vessel on each occasion during the study. The pH remained within acceptable limits during the study (range 7.32 - 7.84).
Dissolved oxygen:
The dissolved oxygen levels of control and test media were measured on the fresh medium remaining in the preparation vessels and on the expired medium in each test vessel on each occasion during the study. Dissolved oxygen levels remained within acceptable limits during the study (range 93 - 103% air saturation value).
Nominal and measured concentrations:
At nominal Tea Tree Oil loading rates of 0.625, 1.25, 2.5, 5, 10 and 20 mg/l, the respective nominal concentrations of terpinen-4-ol concentrations were 0.26, 0.52, 1.03, 2.07, 4.13 and 8.26 mg/l. The mean measured concentrations of terpinen-4-ol at nominal Tea Tree Oil concentrations of 1.25, 2.5, 5, 10 and 20 mg/l were 0.47 (< LOQ, 0.6 mg/l), 1.02, 1.96, 3.94, and 7.46 mg/l, respectively. At the nominal Tea Tree Oil concentration of 0.625 mg/l, the measured concentration of terpinen-4-ol was below the LOD of 0.2 mg/l.

The measured levels of terpinen-4-ol at nominal Tea Tree Oil loading rates of 0.625 and 1.25 mg/l were less than the limit of detection (LOD, 0.2 mg/l) and limit of quantitation (LOQ, 0.6 mg/l) of the analytical method, respectively. As the no effect loading rate was higher than these levels, this had no effect on the validity of the study. At the remaining concentrations, the measured levels of terpinen-4-ol ranged between 92 and 126% of their nominal values in samples of freshly prepared media except at 10 mg/l on Day 1 where the measured concentration was 71% of its nominal value.

In samples of expired (24-hours old) media, the measured concentrations ranged from 76 to 113% of the nominal values (between 70 and 139% of their initial values). The overall mean measured levels of terpinen-4-ol were 0.47 (
In accordance with the recommendation of the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (Number 23), the test results have been expressed in terms of the nominal loading rates (i.e. the nominal weights of the test substance used to prepare the aqueous mixtures from which the WAFs were removed).
Details on test conditions:
- Headspace, fill volume: No headspace, completely filled.
- Aeration: No supplementary aeration was employed.
- Renewal rate of test solution: The control and test media were renewed after 24 hours by transferring the Daphnia to freshly prepared control or test medium.
- No. of organisms per vessel: Twenty Daphnia, four replicates of five animals per vessel, were exposed in each control and test group. The first instar Daphnia were placed at random into completely filled test vessels to give a loading of approximately 26 ml medium per organism.
- Other: No feed was given during the exposure period.

- Chlorine: Free and total chlorine content of the dilution medium were measured before used; they were within acceptable limits.
- Alkalinity: The alkalinity of the dilution medium was measured before use; it was within acceptable limits.
- Culture medium different from test medium: No.

- Photoperiod: A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase.
- Light intensity: The light intensity of the test area is measured periodically; the result of the most recent measurement conducted prior to the start of the definitive test indicated the test area had a light intensity of 535 lux.
- Other: At all concentrations, the test media were colourless.

- Criteria of effect: Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel. The numbers of mobile, immobile and floating Daphnia were counted approximately 24 and 48 hours after the start of the study.

- Range finding study: A range finding test was conducted at nominal loading rates of 0.01, 0.1, 1 and 10 mg/l which was unsuccessful as no immobility was observed. As a result, the test was repeated at a single loading rate of 100 mg/l; all of the Daphnia were immobile at 24 hours. Based on the results of these tests, a main test (originally planned as the definitive test) was conducted at nominal loading rates of 5, 10, 20, 40 and 80 mg/l. However, the pattern of immobility was inconsistent with that seen in the range finding tests; after 48 hours, 75% of the Daphnia at 5 mg/l and all of the animals at 10 mg/l and higher loading rates were immobile. As a result, an additional range finding test was conducted at nominal loading rates of 0.01, 0.1, 1 and 10 mg/l; the results of this test were in line with the main test. Consequently, the definitive test was conducted at nominal loading rates of 0.625, 1.25, 2.5, 5, 10 and 20 mg/l. A dilution medium control group was also included in the test.
Reference substance (positive control):
no
Remarks:
Sensitivity of juvenile Daphnia magna cultured in this lab is periodically assessed using ref. substance potassium dichromate. The most recent result (48-hour EC50 1.3 mg/l), was in line with typical toxicity data for this substance.
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
16.6 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water-accommodated fraction
Basis for effect:
mobility
Remarks on result:
other: 95% CL = 15.3 & 20.0 mg/l
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
13.6 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water-accommodated fraction
Basis for effect:
mobility
Remarks on result:
other: 95% CL = 11.6 & 16.1 mg/l
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water-accommodated fraction
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water-accommodated fraction
Basis for effect:
mobility
Details on results:
- Immobility: Observations of the Daphnia in each control and test vessel made after 24 and 48 hours are listed in Table 2 in 'Any other information on results', and the relationship between immobility and concentration is given in attached Fig. 2. After 48 hours, the lowest loading rate resulting in 100% immobility was 20 mg/l and the highest loading rate at which no immobilisation occurred (the no-observed effect loading rate, NOELR) was 10 mg/l. One of the Daphnia at 2.5 mg/l was immobile after 48 hours; this is not attributed to the test substance as no immobilisation was observed at 5 and 10 mg/l. EL50 and NOELR values were estimated on the basis of these findings.
Reported statistics and error estimates:
Evaluation of data: Statistical analysis was performed using the SAFEStat LD50 application, SAS 8.2 (SAS Institute, 1999). Test results were expressed in terms of the nominal loading rate of Tea Tree Oil. The "no observed effect loading rate" (NOELR) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% is considered to be significant.

Table 2. Cumulative immobilisation.

Nominal loading rate (mg Tea Tree Oil/l)

Cumulative numbers of immobile Daphnia

24 hours

48 hours

R1

R2

R3

R4

total

%

R1

R2

R3

R4

total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

0.625

0

0

0

0

0

0

0

0

0

0

0

0

1.25

0

0

0

0

0

0

0

0

0

0

0

0

2.50

0

0

0

0

0

0

1

0

0

0

1

5*

5.00

0

0

0

0

0

0

0

0

0

0

0

0

10.0

0

0

0

0

0

0

0

0

0

0

0

0

20.0

3

3

5

4

15

75

5

5

5

5

20

100

R : replicate number

* : not considered to be an effect of the test substance

Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EL50 of Tea Tree Oil for the immobilisation of Daphnia magna when tested as water accomodated fractions was 13.6 mg/l (95% confidence limits of 11.6 and 16.1 mg/l). The 'no-observed effect loading rate' of Tea Tree Oil for the immobilisation of Daphnia magna when tested as water accommodated fractions was 10 mg/l.
Executive summary:

This study was conducted in accordance with EC Method C2 and OECD Guideline No. 202 and assessed the acute toxicity of tea tree oil to Daphnia magna under semi-static exposure conditions.  Groups of twenty Daphnia were exposed for 48 hours to Water Accommodated Fractions (WAFs) prepared from aqueous mixtures of tea tree oil, at nominal loading rates of 5, 10 and 20 mg/l or to test media prepared at nominal concentrations of 0.625, 1.25 and 2.5 mg/l by dilution of the WAFs at the higher concentrations.  The test was conducted in completely filled and sealed vessels. Quantification of the test concentrations was based on the measured concentration of terpinen-4-ol.  Based on the measured levels of terpinen-4-ol the overall mean test concentrations ranged between 90 and 99% of their nominal values.  In accordance with the recommendation of the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (Number 23), the test results have been expressed in terms of the nominal loading rates. After 48 hours, the highest loading rate at which no immobilisation occurred (no-observed effect loading rate, NOELR) was 10 mg/l.  The 48-hour EL50 was estimated to be 13.6 mg/l.

Description of key information

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
13.6 mg/L

Additional information

In accordance with the recommendation of the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (Number23), the test results have been expressed in terms of the nominal loading rates (i.e. the nominal weights of the test substance used to prepare the aqueous mixtures from which theWAFs were removed), the EL50.