p-menth-1-en-8-ol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January 2019 – 07 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 10, 18, 32, 56 and 100 mg/L (nominal)
- Sampling method: all concentrations along with the negative control were analyzed for the test item concentration in fresh and old solutions every 24 hour (semi-static test). Aliquouts of solution were taken directly from the aquarium, without fish in the case of fresh solutions.
- Sample storage conditions before analysis: Those solutions not analysed immediately, were kept in refrigerator.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was soluble in the test medium (OECD TG 201 medium) with a mixing protocol carried out before the experimentation, according to the OECD nº23 guidance document on aquatic toxicity testing of difficult substances and mixtures.
The method consisted in stirring each test item nominal concentrations in test medium during 24 hours, in the dark and 20±2ºC prepared by adding directly the test item in flasks containing test medium. For technical feasibility issues, the concentration 3.2 mg/L was diluted from the concentration 320 mg/L. After this step, a settling period of 30 minutes was performed before sampling.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: Not reported
- Length at study initiation (length definition, mean, range and SD): 25 to 30 mm
- Weight at study initiation (mean and range, SD): not specified

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): same as test

FEEDING DURING TEST . None

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

250 mg/L of CaCO3

Test temperature:

22.1 to 23.0ºC

pH:

7.4 to 7.9

Dissolved oxygen:

87 to 99% of air saturation

Nominal and measured concentrations:

Nominal: 0 (control), 10, 18, 32, 56 and 100 mg/L
Measured (geometric mean): 0 (control), 7.7, 13.7, 25.4, 45.6 and 85.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: aquarium
- Material, size, headspace, fill volume: glass, 15cm x 15cm x 20 cm containing 2L of test solution.
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: 1.0 g/L (7 fish / aquarium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Fish test medium (ISO) was prepared as per the OECD guideline 203.
- Intervals of water quality measurement: Temperature, pH and dissolved Oxygen were measured every 24 h in old and fresh solutions.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light / 8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The dead fish were reported every 24 hours, and removed as soon as possible. Fish was considered dead if there was no visible movement (e.g. gill movement) and if touching of the caudal peduncle produced no reaction. The visible abnormalities (loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc…) were reported if need be. Observation at three or six hours after the start of the test was done and checked if need be.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results obtained for the test item gave a LC50-96h between 10 and 100 mg test item/L, value expressed in nominal test item concentration.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

62.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
- Abnormal responses: After 4 hours of exposure, all the fish were dead at 100mg/L therefore this solution was not renewed at 24 h.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The measured concentrations of test item in fresh samples were not maintained within ± 20 per cent of the nominal test item concentrations (-12.3% to -29.4%). According to the results between fresh and old (24 hours), the measured concentrations of the test item in solutions were maintained within ± 20 per cent of the measured initial concentration (0 to -12.4%). Therefore, and in agreement with the Guidance document on aquatic toxicity testing of difficult substances and mixtures OECD nº23, concentrations were expressed in geometric mean measured test item concentrations.

Results with reference substance (positive control):

- Results with reference substance valid? yes (in accordance with laboratory historical data)
- LC50-24 h: 202.4 mg/L [176.7 – 233.0]

Reported statistics and error estimates:

According to the OECD 203, the data obtained are inadequate for the use of the standard methods of calculating the LC50 since no mortality was produced up to 45.6 mg/L and 100% mortality was produced at the next level (highest concentration tested, 85.1 mg/L). Thus, the geometric mean of these two concentrations were used as an approximation for the LC50.

Sublethal observations / clinical signs:

Table 1: Results of test item analyses

Nominal test item concentration   (mg/L)	Results of the determination of test item analysis (mg/L)
	0 hour Initial time	Deviation T0h/Nominal concentration   (%)	24 hours		Deviation T24h fresh /Nominal concentration   (%)	Deviation T0h/T24h old   (%)
			OLD	FRESH
Control	< 1	NA	< 1	< 1	NA	NA
10	8.1	-19.0	7.2	8.0	-20.0	-11.1
18	13.1	-27.2	12.3	15.7	-12.8	-6.1
32	27.0	-15.6	24.3	25.0	-21.9	-10.0
56	46.7	-16.6	43.0	43.9	-21.6	-7.9
100	87.0	-13.0	83.5	NA	NA	-4.0

Nominal test item concentration (mg/L)	Results of the determination of test item analysis (mg/L)
	48 hours		Deviation T48h fresh /Nominal concentration   (%)	Deviation T24h fresh/   T48h old (%)	72 hours		Deviation T72h Fresh /Nominal concentration   (%)	Deviation T48h fresh/T72h old (%)	96 hours	Deviation T72h fresh /T96h old   (%)
	OLD	FRESH			OLD	FRESH			OLD
Control	< 1	< 1	NA	NA	< 1	< 1	NA	NA	< 1	NA
10	7.6	7.9	-21.0	-5.0	7.9	7.7	-23.0	0.0	7.0	-9.1
18	14.9	12.7	-29.4	-5.1	13.1	15.0	-16.7	+3.1	13.2	-12.0
32	21.9	27.6	-13.8	-12.4	27.1	26.1	-18.4	-1.8	24.4	-6.5
56	44.5	47.0	-16.1	+1.4	43.5	49.1	-12.3	-7.4	46.8	-4.7
100	/	/	NA	NA	/	/	NA	NA	/	NA

Nominal test item concentration (mg/L)	Test item analysis (mg/L)								Geometric mean measured (mg/L)
	0 hour Initial time	24 hours		48 hours		72 hours		96 hours
		Old	Fresh	Old	Fresh	Old	Fresh	Old
10	8.1	7.2	8.0	7.6	7.9	7.9	7.7	7.0	7.7
18	13.1	12.3	15.7	14.9	12.7	13.1	15.0	13.2	13.7
32	27.0	24.3	25.0	21.9	27.6	27.1	26.1	24.4	25.4
56	46.7	43.0	43.9	44.5	47.0	43.5	49.1	46.8	45.6
100	87.0	83.5	NA	NA	NA	NA	NA	NA	85.1

Table 2: Fish lethality

Nominal test item concentrations (mg/L)	Geometric mean measured test item concentration   (mg/L)	Number of fish exposed at the start	Cumulative number of dead fish at each observation time (%)
			24h	48h	72h	96h
Control	0	7	0 (0%)	0 (0%)	0 (0%)	0 (0%)
10	7.7	7	0 (0%)	0 (0%)	0 (0%)	0 (0%)
18	13.7	7	0 (0%)	0 (0%)	0 (0%)	0 (0%)
32	25.4	7	0 (0%)	0 (0%)	0 (0%)	0 (0%)
56	45.6	7	0 (0%)	0 (0%)	0 (0%)	0 (0%)
100	85.1	7	7 (100%)	7 (100%)	7 (100%)	7 (100%)

Validity criteria fulfilled:

yes

Remarks:

(mortality in control was 0%; constant conditions were maintained; the dissolved oxygen concentration was within the acceptable range; results were based on geometric mean measured concentrations and were maintained within ± 20%).

Conclusions:

In a short-term toxicity test to Danio rerio (Zebrafish), the 96h-LC50 of the test substance was found to be 62.3 mg/L.

Executive summary:

An acute aquatic toxicity study with Danio rerio (Zebrafish), was conducted on test item according to OECD guideline 203, following GLP. In the previous solubility study the test item was found soluble in ISO fish medium and thus no vehicle was needed. A preliminary range finding study was conducted with test concentrations of 0 (control), 1, 10 and 100 mg/L for a period of 96 h with 3 fish in each group, giving a LC50-96h between 10 and 100 mg test item/L. Based on these results, the definitive test was conducted with nominal test item concentrations of 0 (control), 10, 18, 32, 56 and 100 mg/L (factor of 1.8). As measured concentrations were not maintained within ± 20 per cent of the nominal concentrations, the test was conducted in semi-static conditions with test solutions being renewed every 24 h. A validated analytical method based on GC-FID was used to monitor all test concentrations along with the negative control in fresh and old solutions every 24 hour. The measured concentrations of the test item in solutions were maintained within ± 20 per cent of the measured initial concentration (0 to -12.4%). The geometric mean measured concentrations of each group were 0 (control), 7.7, 13.7, 25.4, 45.6 and 85.1 mg/L. All validity criteria were fulfilled. No mortality was observed up to 45.6 mg/L and 100% mortality (after 4 h exposure) was produced at the next level (highest concentration tested, 85.1 mg/L). Thus, the 96h-LC50

was calculated to be 62.3 mg/L based on the geometric mean of these two concentrations according to the OECD TG 203.

Description of key information

Key study. Test method according to OECD 203, GLP study. The 96h-LC50 of the test substance to Danio rerio was found to be 62.3 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

62.3 mg/L

Additional information

Key study: An acute aquatic toxicity study with Danio rerio (Zebrafish), was conducted on test item according to OECD guideline 203, following GLP.In the previous solubility study the test item was found soluble in ISO fish medium and thus no vehicle was needed.A preliminary range finding study was conducted with test concentrations of 0 (control), 1, 10 and 100 mg/L for a period of 96 h with 3 fish in each group, giving a LC50-96h between 10 and 100 mg test item/L. Based on these results, the definitive test was conducted with nominal test item concentrations of 0 (control), 10, 18, 32, 56 and 100 mg/L (factor of 1.8). As measured concentrations were not maintained within ± 20 per cent of the nominal concentrations, the test was conducted in semi-static conditions with test solutions being renewed every 24 h. A validated analytical method based on GC-FID was used to monitor all test concentrations along with the negative control in fresh and old solutions every 24 hour. The measured concentrations of the test item in solutions were maintained within ± 20 per cent of the measured initial concentration (0 to -12.4%). The geometric mean measured concentrations of each group were 0 (control), 7.7, 13.7, 25.4, 45.6 and 85.1 mg/L. All validity criteria were fulfilled. No mortality was observed up to 45.6 mg/L and 100% mortality (after 4 h exposure) was produced at the next level (highest concentration tested, 85.1 mg/L). Thus, the 96h-LC50 was calculated to be 62.3 mg/L based on the geometric mean of these two concentrations according to the OECD TG 203.