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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:
04 Jun 2017 - 08 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures
Version / remarks:
OECD series on testing and assessment number 23, 2000.
GLP compliance:
yes
Specific details on test material used for the study:
Test item information, batch used for the combined limit/range-finding test and final test 1
Test Facility test item number: 208028/A
Identification: Ylang Ylang I
Appearance: Pale yellow clear liquid
Batch: 16 07 5017 BG
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 09 August 2018 (expiry date)

Test item information, batch used for final test 2
Test Facility test item number: 208028/B
Identification: Ylang Ylang I
Appearance: Clear amber liquid (determined by Charles River Den Bosch)
Batch: 17046299
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2019 (expiry date)

Additional information:
Specific gravity / density: 0.940 (0.933-0.949)
Analytical monitoring:
yes
Remarks:
TOC analysis
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=48 h
Volume: 40 mL
Storage: Samples were stored in a refrigerator (2 - 8°C) until analysis.
At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Instead samples were taken from one vessel of each group.

Additionally, reserve samples of 40 mL were taken for possible analysis. If not 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 batches of Ylang Ylang I tested were UVCBs and not completely soluble in test medium at the loading rates initially prepared. Batch 16 07 5017 BG was described as a pale yellow clear liquid while batch 17046299 was described as a clear amber liquid. No correction was made for the purity/composition of the test item. During preparation of test solutions the test items were treated as being possibly volatile.

Preparation of test solutions started with loading rates individually prepared at concentrations ranging from 0.46 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 through glasswool and microscopically inspected for the presence of undissolved test material. All test solutions prepared at loading rates <10 mg/L were clear and colorless at the end of the preparation procedure. Observations performed on test solutions prepared at loading rates ≥10 mg/L differed between the three tests from being clear and colourless to being slightly hazy. Any residual volumes were discarded.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test, young daphnids with an age of < 24 hours were selected from parental daphnids older than two weeks.

Breeding:
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
Final test 1: The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test
Final test 2: The temperature continuously measured in a temperature control vessel varied between 19 and 21°C during the test
pH:
Final test 1:
pH t=0h : 7.7- 8.0
pH t=48h: 7.4 - 8.0

Final test 2:
pH t=0h : 7.8 - 7.9
pH t=48h: 7.5 - 8.0

Dissolved oxygen:
Final test 1:
O2 t=0h: 8.5- 9.1
O2 t=48h: 4.7 - 7.6

Final test 2:
O2 t=0h: 9.2 - 9.8
O2 t=48h: 4.7 - 9.1
Nominal and measured concentrations:
Nominal concentrations:
Final test 1: WAFs at 10, 18, 32, 56 and 100 mg/L
Final test 2: WAFs at 0.46, 1.0, 2.2, 4.6, 10, 22 and 46 mg/L

Final test 1:
TOC measurements corrected for for the control treatment
Loading rates: control, 10, 18, 32, 56 and 100 mg/L
WAFs at t=0h: n.a, 2.0, 5.9, 8.4,12 and 23 mg TOC/L
WAFs at t=48h n.a, 1.3, 4.1,7.5,11 and 18 mg TOC/L

Final test 2:
TOC measurements corrected for the control treatment
Loading rates: control, 0.46, 1.0, 2.2, 4.6, 10, 22 and 46 mg/L
WAFs at t=0h: n.a, n.q, 0.23, 0.66, 1.6, 3.4, 7.8 and 14 mg TOC/L
WAFs at t=48h n.a, n.q, 0.39, 0.42, 1.1, 2.4, 6.3 and 12 and TOC/L


n.a. – not applicable, n.q. – not quantifiable
Details on test conditions:
Test system:
Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass, closed with no headspace
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 60 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 30 minutes after preparation of the test solutions.

Measurements and Recordings:
Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Combined Limit/Range-Finding Test
The study started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a control and a WAF at 100 mg/L. Test procedure and conditions were similar to those applied in the final tests with the following exceptions:
• Ten daphnids per concentration (two replicates, five daphnids per replicate) were exposed to WAFs at 1.0 and 10 mg/L;
• Dissolved oxygen concentrations and pH were only measured in the control and the WAF at 100 mg/L.

- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
10.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% Cl: 9.4-12 mg/L
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% Cl: 13-18 mg/L
Details on results:
Final test 1:
Measured TOC concentrations showed an increase with the loading rate at the start of the test. At the start of the test all WAFs and the control were observed to be clear and colorless. This did not change during the test period. After 48 hours of exposure >50% of the daphnids exposed to all WAFs were immobilized. These responses were not in agreement with the results of the combined limit/range-finding test and no 48h EL50 could be determined. Therefore, the study was continued with final test 2 using WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10, 22 and 46 mg/L. Test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: ≥ 3 mg/L at the end of the test).

Final test 2:
Measured TOC concentrations showed an increase with the loading rate at the start of the test. At the start of the test WAFs up to and including 4.6 mg/L and the control were observed to be clear and colorless. The WAFs at 10 and 22 mg/L were slightly hazy, while the WAF at 100 mg/L was clear and colorless. This did not change during the test period. After 48 hours of exposure all daphnids exposed to the WAFs at 22 and 46 mg/L were immobilised. No significant immobility was observed in WAFs up to and including 10 mg/L and in the control. The responses recorded in the WAFs at 1.0 and 10 mg/L were in agreement with the results of the combined limit/range-finding test. Therefore, the responses recorded in final test 2 allowed for a reliable determination of EL50 values. Test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test).

Acceptability of the Test: (check with final report)
1. In the control , no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Results with reference substance (positive control):
In conclusion, the actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 24h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.68 mg/L.
The 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L.
Reported statistics and error estimates:
The 24 and 48h-EL50-value were calculated using the Spearman-Karber method (without trim) on the percentages of affected daphnids and the logarithms of the corresponding loading rates of the test item. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Incidence of Immobility in the Final Test 1

Ylang Ylang I, loading rate (mg/L)

Immobility %

t=24h

 

Immobility %

t=48h

Control

0

0

10

15

70

18

55

100

32

85

100

56

100

100

100

100

100


 

Incidence of Immobility in the Final Test 2

Ylang Ylang I, loading rate (mg/L)

Immobility %

t=24h

 

Immobility %

t=48h

Control

0

0

0.46

0

0

1.0

5*

0*

2.2

5*

0*

4.6

0

0

10

0

5

22

85

100

46 100  100 

  *Immobilisation is not an irreversible effect.

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
The 48h-EL50 for Daphnia magna exposed to Ylang Ylang I was 10.4 mg/L based on loading rates (95% confidence interval between 9.4 and 12 mg/L).
Executive summary:

Two full OECDTG 202 GLP tests were performed based on the results of a combined limit/range-finding test. Water Accommodated Fractions (WAFs) of Ylang Ylang I were individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L in final test 1 and 0.46, 1.0, 2.2, 4.6, 10, 22 and 46 mg/L in final test 2 and used as test concentrations. Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to each WAF. The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analyses 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. The studies met the acceptability criteria prescribed by the study plan and were considered valid. The EL50 value was calculated from the combined results of the two final tests. In conclusion, the 48h-EL50 for Daphnia magna exposed to Ylang Ylang I was 10.4 mg/L based on loading rates (95% confidence interval between 9.4 and 12 mg /L).

 

Description of key information

Two full OECDTG 202 GLP tests were performed based on the results of a combined limit/range-finding test. Water Accommodated Fractions (WAFs) of Ylang Ylang I were individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L in final test 1 and 0.46, 1.0, 2.2, 4.6, 10, 22 and 46 mg/L in final test 2 and used as test concentrations. Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to each WAF. The total exposure period was 48 hours and samples for Total Organic Carbon (TOC) analyses 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. The studies met the acceptability criteria prescribed by the study plan and were considered valid. The EL50 value was calculated from the combined results of the two final tests. In conclusion, the 48h-EL50 for Daphnia magna exposed to Ylang Ylang I was 10.4 mg/L based on loading rates (95% confidence interval between 9.4 and 12 mg /L).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
10.4 mg/L

Additional information

The value is based on loading rate: EL50 = 10.4 mg/L