Registration Dossier

Diss Factsheets

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Jun - 11 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 1.06, 2.13, 4.25, 8.50 and 17 mg/L
- Sampling method: Samples were analysed from freshly prepared media after 0, 24, 48 and 72 hours and from corresponding aged test media after 24, 48, 72 and 96 hours.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate volumes of the test item were pipetted directly into the test vessels filled with appropriate volumes of the dilution water. Test media were mixed with an ultraturrax (2 min at 8500 rpm). The test vessels were covered with a floating lid. After a settling period of 2 h the fish were inserted. This procedure was repeated for each renewal day.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium conrol
- Evidence of undissolved material: no, test solutions were clear
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Length at study initiation: 2.53 cm (mean)
- Weight at study initiation: 0.141 g (mean)

ACCLIMATION
- Acclimation period: at least 12 d
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Sera Vipan, Sera GmbH, Heinsberg, Germany
- Feeding frequency during acclimation: 3 time a week (4% of the fish body weight per feeding day); not fed 24 h before study initiation
- Health during acclimation: No mortality was observed during this time. No disease treatments were administered throughout holding and testing.

FEEDING DURING TEST
- None
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
59 mg/L as CaCO3
Test temperature:
22.9 - 23.9 °C (mean: 23.3 °C ± 0.3 °C)
pH:
6.97 - 7.57
Dissolved oxygen:
86 - 100% O2 saturation
Nominal and measured concentrations:
nominal: control, 1.06, 2.13, 4.25, 8.50 and 17 mg/L
geometric mean measured: < LOQ, 0.301, 0.462, 1.26, 3.10, 9.30 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria
- Type (delete if not applicable): covered with a floating lid
- Material, size, headspace, fill volume: Glass, 13 L (25.5/18/28 cm), headspace: 3 L, fill volume: 10 L
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): every 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.099 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water of local origin
- Hardness: 10 - 250 mg CaCO3/L
- Conductivity: 168 µS/cm
- Acidity: 0.10 mmol/L
- Acid capacity: 0.60 mmol/L
- Culture medium different from test medium: same as test
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all test vessels at the beginning and at the end of the exposure and every 24 h from fresh and old test media. Water temperature was recorded once per hour with a data logger. Light intensity was measured on the water surface at test start.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: 7 - 750 lux

EFFECT PARAMETERS MEASURED
- Mortality was recorded after 2, 24, 48, 72 and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: control, 17 mg/L
- Results used to determine the conditions for the definitive study: 100% mortality was recorded after 24 h at the limit concentration.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
6.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% CI
Remarks:
3.10 - 9.30 mg/L
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 9.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: Up to a concentration of 3.1 mg/L all fish behaved normal. At the highest test concentration surviving fish were observed to be lethargic, staggering, on a side position and/or missing escape reflex.
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, test solutions were clear
Reported statistics and error estimates:
LC50 value was calculated by linear regression.
Sublethal observations / clinical signs:

Table 1: Cumulative mortality

Geometric mean measured test item concentrations [mg/L]

Cumulative mortality [%] during the study [h]

2

24

48

72

96

9.30

0

0

29

71

71

3.10

0

0

0

0

0

1.26

0

0

0

0

0

0.452

0

0

0

0

0

0.301

0

0

0

0

0

Control

0

0

0

0

0

Table 2: Measured concentrations of the test substance

Nominal concentration

0 h (fresh)

24 h (old)

24 (fresh)

48 h (old)

48 h (fresh)

72 h (old)

72 h (fresh)

96 h (old)

Geometric mean [mg/L]

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

17

11.8

69

9.10

54

14.6

86

11.2

66

9.85

58

9.26

54

5.05

30

6.91

41

9.30

8.5

4.97

58

3.23

38

2.58

30

2.13

25

3.68

43

2.55

30

3.29

39

3.15

37

3.10

4.25

2.56

60

1.23

29

1.53

36

1.09

26

1.17

28

0.810

19

1.24

29

1.01

24

1.26

2.13

0.833

39

0.345

16

0.827

39

0.480

23

0.291

14

0.187

9

0.584

27

0.477

22

0.452

1.06

0.587

55

0.231

22

0.371

35

0.157

15

0.498

47

0.296

28

0.256

24

0.223

21

0.301

Control

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

Table 3: Measured concentrations of the metabolite

Nominal concentration

0 h (fresh)

24 h (old)

24 (fresh)

48 h (old)

48 h (fresh)

72 h (old)

72 h (fresh)

96 h (old)

Geometric mean [mg/L]

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

17

< LOQ

<1

0.790

6

0.0397

<1

0.599

5

0.0425

<1

0.619

5

0.0383

<1

0.142

1

0.203

8.5

0.0955

1

1.06

16

0.00953

<1

0.349

5

0.0434

<1

0.684

10

0.0192

<1

0.285

4

0.157

4.25

0.00393

<1

0.236

7

0.0166

<1

0.157

5

0.0122

<1

0.214

6

0.0128

<1

0.170

5

0.0787

2.13

< LOQ

<1

0.150

9

0.0222

1

0.199

12

0.00546

<1

0.102

6

0.0101

<1

0.130

8

0.0460

1.06

0.00836

1

0.0786

9

0.0214

3

0.1253

15

0.00791

1

0.0998

12

0.00999

1

0.0792

10

0.0406

Control

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

Validity criteria fulfilled:
yes
Conclusions:
This short-term study with fish resulted in a LC50 (96 h) of 6.72 mg/L (geom. mean measured concentration).
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 Jul - 17 Jul 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
No details were provided.
Vehicle:
no
Details on test solutions:
The test item was pestled in a mortar, weighed into water and centrifuged in an Ultra-Turrax to create the test solutions. At all concentrations tested, a certain amount of the test item remained undissolved and was floating on the surface of the test solutions. Therefore, the actual test concentrations were measured daily using gas chromatography.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Common name: Brachydanio rerio
Date of birth: 1992-04-18
Date of shipment: 1992-06-10
Length at study initiation: 2.5 - 3.5 cm
Mortalita during acclimatisation: <2%
Medical pretreatment: None
Water for acclimatisation: Synthetic freshwater (according to ISO)


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
14.1 °dH
Test temperature:
21.4 - 22.5 °C; A small decrease in test temperatures during the study period (about 1 °C in most cases) was observed in most of the experiments.
pH:
7.1 - 8.0
Dissolved oxygen:
8.1 - 10.4 mg/L (O2 saturation: 93.4 - 120.0%)
Nominal and measured concentrations:
nominal: control, 7.8, 11, 16, 22, and 31 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: test aquaria with 300 x 135 x 200 mm, containing 5 L test medium
- Aeration: Yes

TEST MEDIUM / WATER PARAMETERS
- Dilution water: Synthetic freshwater (ISO)
- Controls: Synthetic freshwater (ISO)
- Solubilizing aid: Treatment with an Ultra-Turrax

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h darkness per day

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)

Assesessed during the experiment at 0, 24, 48, 72, and 96 h:
- Abiotic parameters: Temperature (°C), dissolved oxygen (mg/L), oxygen saturation (% ), pH
- Outer appearance and behaviour of the test animals
- Mortality

Assesessed after death during the experiment or sacrification after termination of the experiment:
- Body weight and length of the test animals

TEST CONCENTRATIONS
- Nominal test concentrations: 7.8, 11, 16, 22, and 31 mg/L
- Actual test concentrations were measured daily using gas chromatography
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
13.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
15.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality (fish)
Remarks on result:
other: as geom. mean of LC0 and LC100 as determined
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
18.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
mortality (fish)
Sublethal observations / clinical signs:

Table 1 Abiotic parameters

Control

Time (h)

0

24

48

72

96

Temperature (°C)

21.5

21.4

21.1

21.1

21.0

Dissolved oxygen (mg/L)

8.6

8.3

9.4

8.1

9.9

Oxygen saturation (%)

99.9

96.3

108.4

93.4

114.1

pH

7.9

7.8

7.1

7.7

7.7

at 7.8 mg/L

Time (h)

0

24

48

72

96

Temperature (°C)

22.3

21.4

21.1

21.1

21.2

Dissolved oxygen (mg/L)

8.7

8.4

10.2

8.7

8.8

Oxygen saturation (%)

102.5

97.4

117.6

100.3

101.7

pH

7.9

7.5

7.4

7.3

7.4

at 11 mg/L

Time (h)

0

24

48

72

96

Temperature (°C)

22.4

21.5

21.1

21.1

21.3

Dissolved oxygen (mg/L)

8.6

8.5

10.4

8.8

8.7

Oxygen saturation (%)

101.5

98.7

120.0

101.5

100.7

pH

8.0

7.5

7.6

7.3

7.5

at 16 mg/L

Time (h)

0

24

48

72

96

Temperature (°C)

22.3

21.5

21.2

21.1

21.4

Dissolved oxygen (mg/L)

8.6

8.6

10.3

8.7

8.5

Oxygen saturation (%)

101.3

99.9

119.1

100.3

98.6

pH

7.9

7.6

7.4

7.4

7.5

at 22 mg/L

Time (h)

0

24

48

72

96

Temperature (°C)

22.5

21.5

21.2

21.2

NA

Dissolved oxygen (mg/L)

8.5

8.7

10.6

8.9

NA

Oxygen saturation (%)

100.5

101.0

122.5

102.9

NA

pH

7.8

7.6

7.7

7.6

NA

at 31 mg/L

Time (h)

0

24

48

72

96

Temperature (°C)

22.4

21.3

NA

NA

NA

Dissolved oxygen (mg/L)

8.4

8.4

NA

NA

NA

Oxygen saturation (%)

99.2

97.2

NA

NA

NA

NA = not assessed

Table 2. Mortality

 No mortality within 96 h was observed in control animals and at concentrations of the test item of 7.8, 11, and 16 mg/L.

At 22 mg/L

Time (h)

2

24

48

72

96

absolute number of deaths

0

5

0

5

NA

cumulative number of deaths

0

5

5

10

NA

cumulative number of deaths (%)

0

50

50

100

NA

at 31 mg/L

Time (h)

2

24

48

72

96

absolute number of deaths

10

NA

NA

NA

NA

cumulative number of deaths

10

NA

NA

NA

NA

cumulative number of deaths (%)

100

NA

NA

NA

NA

NA = not assessed

Table 3: Outer appearance and behaviour

at 11 mg/L

after 2 h

10 animals

Sluggish swimming

after 24 h

10 animals

Sluggish swimming

after 48 h

10 animals

Sluggish swimming

after 72 h

10 animals

Sluggish swimming

at 16 mg/L

after 2 h

10 animals

Sluggish swimming

after 24 h

10 animals

Sluggish swimming

after 48 h

8 animals

Sluggish, lethargic swimming

2 animals

Sluggish, lethargic swimming, abnormal posture

after 72 h

8 animals

Sluggish, lethargic swimming

2 animals

Sluggish, lethargic swimming, abnormal posture

after 96 h

10 animals

Sluggish, lethargic swimming

at 22 mg/L

after 2 h

10 animals

Akinetic, uncontrolled swimming movements upon touching

after 24 h

5 animals

Akinetic, uncontrolled swimming movements upon touching

after 48 h

5 animals

Akinetic, uncontrolled swimming movements upon touching

Validity criteria fulfilled:
yes
Conclusions:
In this short-term toxicity test in Brachydanio rerio, the non-lethal concentration 0% (LC0) and the lethal concentration 100% (LC100) were determined as 13.2 and 18.4 mg/L, respectively. The geometric mean between both values (LC50) was calculated as 15.6 mg/L.
Executive summary:

The short-term toxicity of the test item, Menthol H&R Komp., was assessed in a test in Brachydanio rerio. The test was conducted according to the EU method No. C.1. The test animals were exposed to the test at concentrations of the test item 7.8, 11, 16, 22, and 31 mg/L for up to 96 h and were observed for mortality as well as for changes in their outer appearance and behaviour. Body weight and length of the fish were also recorded. From the mortality data obtained in this study, values for the lethal concentration 0% (LC0) and the lethal concentration 100% (LC100) were determined as 13.2 and 18.4 mg/L, respectively. The geometric mean between both values was calculated as 15.6 mg/L.

Description of key information

LC50 (96 h) = 6.72 mg/L (measured, OECD 203); read-across

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
6.72 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 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 conducted according to OECD 203 (GLP). Danio rerio was exposed to nominal concentrations of 1.06, 2.13, 4.25, 8.50 and 17 mg/L for 96 h under semi-static conditions. The exposure concentrations were verified by GC/MS analysis. Measurements indicate that the measured concentrations did not remain within 80-120% of nominal. The recovery rate ranged from 14 - 86% in the freshly prepared media and 9 - 66% in the aged test solutions. Thus, the effect concentrations were based on the geometric mean of measured concentrations. After 96 h an LC50 of 6.72 mg/L was calculated by regression analysis.
The supporting study with L-menthol, performed according to EU Method C.1 (GLP), resulted in a slightly higher LC50 (96 h) of 15.6 mg/L (measured).

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 fish.