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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jan 2018 - 14 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Ylang Ylang III
Appearance: Pale yellow clear liquid
Batch: 17106567
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2019 (expiry date)

Additional information
Test Facility test item number: 208293/C
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name: Ylang Ylang III oil
CAS number: 83863-30-3 (8006-81-3)
Irritant or corrosive: Yes
Specific gravity / density: 0.914
Analytical monitoring:
yes
Remarks:
TOC to confirm WAF procedure
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h and t=96 h
Volume 40 mL
Storage Samples were stored in a refrigerator (2-8°C) until analysis

Additionally, reserve samples of 40 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a refrigerator (2-8°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
The batch of Ylang Ylang III tested was a pale yellow clear liquid. The test item was a UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. During preparation of test solutions the test item was treated as being possibly volatile. Preparation of test solutions started with loading rates individually prepared at concentrations ranging from 0.10 to 100 mg/L. A two-day period of magnetic stirring in closed vessels with minimal headspace in the dark was applied to ensure maximum dissolution of the test item in medium. The obtained aqueous mixtures were allowed to settle overnight. Thereafter, the Water Accommodated Fractions (WAFs) were collected by means of siphoning and microscopically inspected for the presence of undissolved test material. Any residual volumes were discarded.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length*: Range-finding test: 3.1 ± 0.4 cm cm, Final test: 2.5 ± 0.1 cm
Mean weight*: Range-finding test: 0.51 ± 0.21 g, Final test: : 0.17 ± 0.03 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 54

* Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

ACCLIMATION
Quarantine/Acclimatisation: At least 12 days after delivery
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel- Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality: parameters Were kept within the optimum limits for the respective fish species.
Feeding: Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands)
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

FEEDING DURING TEST
No feeding from 24 hours prior to the test and during the total test period
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
180 mg CaCO3 per litre
Test temperature:
20-24°C
pH:
Start of test (0 hr): 7.8- 8.0
End of test (96hr): 7.3 - 7.6
Dissolved oxygen:
Start of test (0 hr): 8.6 - 9.2
End of test (96hr): 4.8 - 6.2
Nominal and measured concentrations:
Nominal: WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L.

Measured TOC concentrations corrected for the control treatment
Loading rate (mg/L): control, 4.6, 10, 22, 46 and 100 mg/L.
TOC (t=0hr): n.a, n.a, 0.48, 1.6, 4.1 and 7.1 mg/L
TOC (t=96hr): n.a, n.a, 0.18, 0.78, n.a and n.a mg/L

n.a. – not applicable
Details on test conditions:
TEST SYSTEM
- Test duration: 96 hours
- Test type: Static
- Test vessels: 3.2 litres, all-glass, closed with no headspace
- Number of fish: 7 per concentration
- Loading: 0.37 g fish/Litre, i.e. 7 fish per 3.2 litres of test medium
- Aeration: The test media were not aerated during the test.
- Introduction of fish: Within 30 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 unit and 1.0°C.

TEST MEDIUM / WATER PARAMETERS
- Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
- Intervals of water quality measurement: dissolved oxygen content, pH and temperature were recorded daily in all vessels with surviving fish beginning at the start of the test (day 0).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Illumination: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality and other effects: At 2¾, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dead fish were removed when observed

TERMINAL PROCEDURES
Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study : was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three fish per concentration were exposed to WAFs prepared at 0.10, 1.0, 10 and 100 mg/L.
• Dissolved oxygen concentrations, pH and temperature were only measured in the lowest and the highest test concentration with surviving fish.
• Additional samples for TOC analysis were taken from the WAF at 100 mg/L when all fish had died after 24 hours of exposure.

- Results used to determine the conditions for the definitive study: yes. All fish exposed to the WAF at 100 mg/L died within 24 hours. No mortality was observed at any of the lower WAFs during the test period. The expected LL50 was between WAFs prepared at loading rates of 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
pentachlorophenol (PCP)
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95%-confidence interval (22 -46 mg/L)
Details on results:
Measured Concentrations:
It should be noted that concentrations below 1.0 mg TOC/L are only indicative. Nevertheless, an increase with the loading rate at the start of the test was observed.
Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared.

Mortality and Other Effects:
All fish exposed to the WAFs at 46 mg/L and higher died within 24 hours. No mortality was observed at any of the lower WAFs during the test period.

Acceptability of the Test
1. No mortality was observed in the control at the end of the test.
2. Test conditions were maintained constant throughout the test.
3. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout the test (>5 mg/L at 22°C), except for a slight deviation in the WAF at 22 mg/L at the end of the test.
4.No specific analysis on water samples was performed. Effects were based on loading rates.

Oxygen concentration in the WAF at 22 mg/L was 4.8 mg/L at the end of the test and therewith just below 60% of the air saturation value. No mortality or clinical effects were observed at this WAF at the end of the test.
Results with reference substance (positive control):
Under the conditions of the present test, all carp exposed to a PCP concentration of 0.46 mg/L died within 24 hours, while no mortality occurred at 0.10 and 0.22 mg/L. The 96h-LC50 for carp exposed to PCP was 0.32 mg/L (95 % confidence interval between 0.22 and 0.46 mg/L). This effect was already reached within 24 hours of exposure.

The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.
Reported statistics and error estimates:
The LL50 could not be determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding loading rates. This was because there was no concentration between the highest loading rate (A) at which 0% mortality and the lowest loading rate (B) at which 100% mortality occurred. Instead, the LL50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.

Incidence of Mortality and Total Mortality during the Final Test

Ylang Ylang III,
Loading rate (mg/L)

Initial
Number
of fish

Cumulative mortality

Total

Mortality (%)

2¾h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

4.6

7

0

0

0

0

0

0

10

7

0

0

0

0

0

0

22

7

0

0

0

0

0

0

46

7

0

7

7

7

7

100

100

7

3

7

7

7

7

100

 

Clinical Effects Observed during the Final Test


Ylang Ylang III
Loading rate (mg/L)

Time of
recording
(hours)

Specification of effects

Relative
number

Control, 4.6, 10

 

22

 

 

 

46

 

 

100

2¾-96

 

2¾-24

48

72-96

 

 

 

 

No abnormalities

 

No abnormalities

Swimming at the surface

No abnormalities

 

Immobile

Loss of equilibrium

 

Immobile

7/7

 

7/7

7/7

7/7

 

3/7

4/7

 

4/4


Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
In conclusion, the 96h-LL50 for carp exposed to Ylang Ylang III was 32 mg/L based on loading rates (95% confidence interval between 22 and 46 mg/L) and was already reached after 24 hours of exposure.
Executive summary:

An evaluation of acute toxic effects of Ylang Ylang III in Cyprinus carpio was investigated in accordance to GLP and OECD 203 guidelines. Based on the results of a preceding range-finding test, Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L and used as test concentrations. Seven fish per group were exposed to a control and to the WAFs under static conditions.The total exposure period was 96 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of the test. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to a minimum. TOC-analysis is a non-specific method and the test substance is a UVCB, therefore the effect parameters were reported in terms of loading rates initially prepared. All fish exposed to the WAFs at 46 mg/L and higher died within 24 hours. No mortality was observed at any of the lower WAFs during the test period. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 96h-LL50 for carp exposed to Ylang Ylang III was 32 mg/L based on loading rates (95% confidence interval between 22 and 46 mg/L) and was already reached after 24 hours of exposure.

Description of key information

An evaluation of acute toxic effects of Ylang Ylang III in Cyprinus carpio was investigated in accordance to GLP and OECD 203 guidelines. Based on the results of a preceding range-finding test, Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L and used as test concentrations. Seven fish per group were exposed to a control and to the WAFs under static conditions.The total exposure period was 96 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of the test. Due to the potential volatile nature of the test item, the exposure was performed in airtight closed vessels with headspace reduced to a minimum. TOC-analysis is a non-specific method and the test substance is a UVCB, therefore the effect parameters were reported in terms of loading rates initially prepared. All fish exposed to the WAFs at 46 mg/L and higher died within 24 hours. No mortality was observed at any of the lower WAFs during the test period. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 96h-LL50 for carp exposed to Ylang Ylang III was 32 mg/L based on loading rates (95% confidence interval between 22 and 46 mg/L) and was already reached after 24 hours of exposure.

Key value for chemical safety assessment

LC50 for freshwater fish:
32 mg/L

Additional information

Based on loading rate.