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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was carried out according to 92/69/EEC method C.2 (1992) and GLP, only four concentrations tested, TOC analysis
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
from 1992
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal concentrations of 2.6, 5.3, 10.5 and 21 mg/L
- Sampling method: not reported
- Sample storage conditions before analysis: not reported
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was weighed into water, treated for 60 seconds at 8000 rpm with an ultra-turrax and afterwards stirred for 24 hours an a magnetic stirrer.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): To remove undissolved particles, the resulting emulsion was filtered using folded filter of the pore size 7-12 µm
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: Laboratory of Bayer AG Leverkusen, cloned cloned at the Federal Health Office (BGA) in Berlin, Germany
- Age at study initiation (mean and range, SD): 0 - 24 hours
- Weight at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD): not reported
- Method of breeding: kept in M4-Medium according to Elendt and BGA. (1992)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
15.2 °dH
Test temperature:
19.9-20.2 °C at 48 h
pH:
7.6 - 7.9 at 48 h
Dissolved oxygen:
8.1 - 8.8 at 48 h
Nominal and measured concentrations:
Nominal: 2.6, 5.3, 10.5, 21 mg/L
Measured, start of test: 2, 4, 8, 15 mg/L (refers to TOC determination. 1 mg/L TOC equals to 1.4 mg/L of the test substance. Measured test substance concentrations are 2.8, 5.6, 11.2, 21 mg/L)
Measured, 48 h: 2, 3, 6, 9 mg/L (refers to TOC determination. 1 mg/L TOC equals to 1.4 mg/L of the test substance. Measured test substance concentrations are 2.8, 4.2, 8.4, 12.6 mg/L)
Details on test conditions:
TEST SYSTEM
- Test vessel: due to loss of substance in the pre-tests, closed bottles were used (so-called "Karlsruhe Flaschen")
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: material and size not reported, 250 - 300 mL test solution and undefined headspace
- Aeration: not ventilated
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- no details on test medium reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light/8 hours dark
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : number of immobile daphnids after 24 hours and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not reported
- Range finding study: no results reported
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
4.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
16.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
9.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: calculated as geometric mean of EC0 and EC100
Details on results:
No additional information is given.
Results with reference substance (positive control):
No reference substance was tested.
Validity criteria fulfilled:
yes
Conclusions:
The EC0 based on immobilisation of test animals was 4.9 mg/L (after 48 hours) and the EC100 was 16.9 mg/L (after 48 hours) in a static acute toxicity test performed for 48 hours with daphnids. The EC50 of 9.1 mg/L was calculated as geometric mean of the EC0 and EC100.
Executive summary:

An acute toxicity study with the test substance was conducted over a period of 48 hours in a static system in accordance with EU method C.2 and under GLP conditions. 10 Daphnia magna STRAUS parthenogenetic females aged 0 to 24 hours were used per test vessel. The test solutions at nominal test concentrations of 2.6, 5.3, 10.5, 21 mg/L were prepared by dissolving the test substance with the help of a stirrer in M4-medium, which was prepared according to Elendt. Due to the loss of substance in pre-tests the study was performed in closed systems. The nominal concentrations were confirmed by measuring the TOC content at the beginning of the test and after 48 hours. The determination of the actual concentrations by analysis of the TOC revealed measured concentrations in the range from 60 to 107% of the nominal concentrations. The tests were performed at a temperature of about 20 °C at pH 7.6 to 7.9. The dissolved oxygen concentration was between 8.1 and 8.8 mg/L. The photoperiod was 16 hours light and 8 hours dark. The EC0 and EC100 values were determined directly from the study results by considering the arithemetic mean of the concentrations measured after 0 and 48 hours. The EC0 based on immobilisation of test animals was 4.9 mg/L (after 48 hours) and the EC100 was 16.9 mg/L (after 48 hours). The EC50 of 9.1 mg/L was calculated as geometric mean of the EC0 and EC100.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Oct 12, 2002 - Dec 12, 2002
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:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Principles of method if other than guideline:
no deviations
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
yes
Details on sampling:
no data
Vehicle:
no
Details on test solutions:
Before the beginning of the study, the test substance was pulverized. A stock solution was prepared by adding 119.9 mg of the test item to 1 L of dilution water, followed by treatment in an ultrasonic bath for 1 h and subsequent stirring on a magnetic stirrer for a further 24 h. Undissolved particles of the test item were then removed by filtration using a folded filter with a pore size of 7-12 µm.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: Strauss
- Source: Bundesgesundheitsamt [Federal Health Office] (Berlin, Germany)
- Method of initiation: Neonates were placed in prepared media
- Method of administration: Stock solution
- Medium renewal: None
- Feeding during test: None

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
not applicable
Hardness:
13.8 dH (=246.3 mg/L CaCO3)
Test temperature:
mean ±1 °C (in the temperature range 18-22 °C)

Please, see section "Any other information on materials and methods incl. tables" for a tabulated listing of the temperatures in each experimental setting at 48 h.
pH:
Please, see section "Any other information on materials and methods incl. tables" for a tabulated listing of pH values prevailing in each experimental setting at 48 h.
Dissolved oxygen:
Please, see section "Any other information on materials and methods incl. tables" for a tabulated listing of the values for the amount of dissolved oxygen (mg/L) and oxygen saturation (%) in each experimental setting at 48 h.
Salinity:
no data
Nominal and measured concentrations:
Nominal test concentrations: 3.2, 6.3, 12.5, 25, 50, and 100 mg/L

Measured concentrations ranged from 93.1-104% of nominal values at 0 h, and from 81.3 - 91.6% of nominal values at 48 h.
Please, see section "Any other information on materials and methods incl. tables" for a tabulated listing of the actual concentrations of the test item in each experimental setting at 0 and 48 h.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mg glass beakers
- Type: open
- Fill volume: 20 mL of test medium
- Aeration: None
- Medium renewal: None
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted water (so-called "M4 medium'", originally described in Water Research 24:1157-67), prepared according to the recommendations of the Bundesgesundheitsamt [Federal Health Office] (Berlin, Germany). This standard dilution water was used for both, the maintenance of the test animals and the preparation of stock and test solutions of the test substance.


OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h darkness per d
- Light intensity: <1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Alterations of the normal mobility and loss of locomotory actions, at 24 and 48 h

TEST CONCENTRATIONS
- Nominal test concentrations: 3.2, 6.3, 12.5, 25, 50, and 100 mg/L
- Results used to determine the conditions for the definitive study: GC analysis (see section "Details on analytical methods")
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
11.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
26.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 14.7 - 48.2 mg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
92.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Please, see the section "any other information on results incl. tables" for a tabulated listing of the degree of immobilisation observed in the test animals at different concentrations of the test item and the two images attached below for graphical representations of the dose-response-relationship observed at 24 and 48 h, respectively.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
no data

Immobilisation of Daphnia magna after an exposure of 24 h

Immobilisation of Daphnia magna

(initial population: 20)

0 mg/L

3.2 mg/L

6.3 mg/L

12.5 mg/L

25 mg/L

50 mg/L

100 mg/L

Absolute

0

0

0

0

0

4

16

Cumulative

0

0

0

0

0

4

16

Cumulative (%)

0

0

0

0

0

20

80

Immobilisation of Daphnia magna after an exposure of 48 h

Immobilisation ofDaphnia magna

(initial population: 20)

0 mg/L

3.2 mg/L

6.3 mg/L

12.5 mg/L

25 mg/L

50 mg/L

100 mg/L

Absolute

0

0

0

0

4

2

20

Cumulative

0

0

0

0

4

18

20

Cumulative (%)

0

0

0

0

20

90

100

Validity criteria fulfilled:
not specified
Conclusions:
In this acute toxicity study in Daphnia magna, the effective concentration 0% (EC0) and the effective concentration 100% (EC100) for immobilisation induced by an exposure to the test item for 24 h were determined as 25 and 100 mg/L, respectively. For an exposure period of 48 h, the respective values were 11.35 and 92.35 mg/L. Furthermore, effective concentration 50% (EC50) values of 37.7 and 26.6 mg/L were determined for the 24 h and the 48 h exposure, respectively.
Executive summary:

The acute toxicity of the test item, Menthol L H&R, was tested in an immobilisation assay in Daphnia magna. The assay was conducted according to EU method C.2. The test subjects were exposed to Menthol L H&R at nominal concentrations of 3.2, 6.3, 12.5, 25, 50, and 100 mg/L for up to 48 h. Test subjects were observed for alterations of the normal mobility behaviour and loss of locomotory activity. From the data obtained through these observations, the effective concentration 0% (EC0) and the effective concentration 100% (EC100) for immobilisation induced by an exposure to the test item for 24 h were determined as 25 and 100 mg/L, respectively. For an exposure period of 48 h, the respective values were 11.35 and 92.35 mg/L. Furthermore, effective concentration 50% (EC50) values of 37.7 and 26.6 mg/L were determined for the 24 h and the 48 h exposure, respectively.

Description of key information

EC50 (48 h) = 9.1 mg/L (measured, EU Method C.2); read-across

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
9.1 mg/L

Additional information

Since no data for the target substance (L-menthyl acetate, CAS 2623-23-6) are available, the aquatic toxicity is assessed on the basis of data from one structurally similar substance (menthyl acetate, CAS 89-48-5) and a possible product of abiotic hydrolysis (L-menthol, CAS 2216-51-5). A detailed justification of the analogue approach is provided in the overall summary and in the technical dossier in IUCLID Section 13.

The key study with the source substance menthyl acetate was performed according to EU Method C.2 (GLP). Daphnia magna was exposed to nominal concentrations of 2.6, 5.3, 10.5 and 21 mg/L for 48 h under static exposure conditions in a closed system due to volatility of the substance. Exposure concentrations very verified by TOC analysis with a recovery rate of 60 - 107% of nominal. Immobile Daphnids were counted after 24 and 48 h. After 48 h an EC50 of 9.1 mg/L was calculated as the geometric mean of EC0 (4.9 mg/L) and EC100 (16.8 mg/L). All exposure concentrations were based on the arithmetic mean of measured concentrations.
The supporting study with L-menthol, performed according to EU Method C.2 (GLP), resulted in a slightly higher EC50 (48 h) of 26.6 mg/L (nominal).

Based on the results from one structurally related source substance and the expected hydrolysis product (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) it can be concluded that L-menthyl acetate has toxic effects on freshwater invertebrates.