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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:
26 May 2017 - 30 Oct 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:
Adopted Apr 13, 2004
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Copaiba balsam oil
Appearance: Pale yellow liquid
Batch: S-71699
Purity/Composition:UVCB
Test item storage: At room temperature protected from light
Stable under storage conditions until: 14 February 2019 (expiry date)
Analytical monitoring:
yes
Remarks:
TOC analysis for WAF preparation
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 50 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 50 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
Remarks:
Water Accomodated Fractions (WAFs) were used
Details on test solutions:
Preparation of test solutions started with loading rates individually prepared ranging between 1.0 and 100 mg/L in the combined limit/range-finding test and between 0.46 and 10 mg/L in the range-finding and final test. A 2-day period of magnetic stirring in closed vessels with minimal headspace and in the dark was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. For the final test the lower concentrations were prepared as a series of dilution of the WAF prepared at a loading rate of 0.46 mg/L in test medium. Microscopic observation of WAFs showed that they did not contain undissolved test material.

The final test solutions were all clear and colourless, except the WAF prepared at 10 mg/L in the range-finding test, which was observed to be slightly hazy. It should be noted that the WAF prepared at a loading rate of 10 mg/L in the final test was not used for the exposure. It was used only for TOC analysis that should confirm proper preparation of WAFs. Any residual volumes were discarded.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test system:
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:
The temperature continuously measured in a temperature control vessel was 20°C during the test.
pH:
pH t=0h : 7.8-7.9
pH t=48h: 7.6-7.7
Dissolved oxygen:
O2 t=0h : 8.4-8.5
O2 t=48h: 8.6-8.8
Nominal and measured concentrations:
Nominal loading rates: Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 0.46 and 1.0 mg/L and used as test concentrations. Solutions containing 4.6, 10, 22 and 46% of the lower WAF were prepared and used as lower test concentrations.

TOC measurements corrected for controls:
Loading rates (mg/L): Controls, 0,46 mg/L, 1.0 mg/ L, 10 mg/L
WAFs: t=0h: n.a, 0.012, n.a, 0.49 mg TOC/L
WAFs: t=48h n.a, n.a, n.a,n.a, mg TOC/L

The WAF of 10 mg/L was prepared only to show the increasing trend during the preparation procedure and not for the exposure.
Details on test conditions:
Test Concentrations:
Copaiba balsam oil: WAFs prepared at 0.46, 1.0 mg/L and solutions containing 4.6, 10, 22 and 46% of the WAF prepared at a loading rate of 0.46 mg/L
Controls: Test medium without test item or other additives

Test Procedure and Conditions:
Test duration : 48 hours
Test type: Static
Test vessels: 60 mL, all-glass, airtight closed with headspace reduced to minimum
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 36 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.

Range-Finding Test
Because a high immobilisation rate was observed in all WAFs tested in the combined limit/range-finding test, an additional range-finding test was performed. Test procedure and conditions were similar to those applied in the final test with the following exceptions
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to WAFs prepared at loading rates of 0.46, 1.0 and 10 mg/L and to a control.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration





Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL 0.29-0.37 mg/L
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
46% of WAF prepared at 0.46 mg/L
Details on results:
Copaiba balsam oil proved to be very poorly soluble in test medium as only 3.4-6.2% of the nominal TOC concentrations were measured at the start of the test. TOC content of the pure substance was 78.99%. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. No immobility was observed in the control and the four lowest concentrations tested during the exposure period. In the WAF prepared at 0.46 mg/L no daphnids were immobilised after the first 24 hours of exposure, whereas 95% was observed to be immobilised at the end of the test. In the highest concentration, 50% daphnids were immobilised after the first 24 hours of exposure, whereas total immobilisation was observed at the end of the exposure.

Acceptability of the Test:
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.

Experimental Conditions:
The results of measurement of pH and oxygen concentrations (mg/L) 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). The temperature continuously measured in a temperature control vessel was 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).
Results with reference substance (positive control):
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-EC50-values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item loading rate using the maximum likelihood estimation method. It should be noted that the solutions containing 4.6, 10, 22 and 46% of the WAF prepared at a loading rate of 0.46 mg/L were treated as a loading rates of 0.022, 0.046, 0.10 and 0.22 mg/L, respectively. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)

Replicate

Copaiba balsam oil

% of WAF prep. at a loading rate of 0.46 mg/L

WAF prep. at loading rate (mg/L)

Control

4.6

10

22

46

0.46

1.0

0

A

5

5

5

5

5

5

5

B

5

5

5

5

5

5

5

C

5

5

5

5

5

5

5

D

5

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

20

 

24

A

0

0

0

0

0

0

3 (5)

B

0

0

0

0

0

0

2 (3)

C

0

0

0

0

0

0

2 (5)

D

0

0

0

0

0

0

3 (5)

Total immobilised

0

0

0

0

0

0

10

Effect %

0

0

0

0

0

0

50

 

 

 

 

 

 

 

48

A

0

0

0

0

0

5

5 (1)

B

0

0

0

0

0

5

5 (1)

C

0

0

0

0

0

5#

5

D

0

0

0

0

0

4

5 (2)

Total immobilised

0

0

0

0

0

19

20

Effect %

0

0

0

0

0

95

100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility,#Microscopic observation revealed no test item attached to the daphnids,

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
The 48h-EL50 for Daphnia magna exposed to Copaiba balsam oil was 0.33 mg/L (95% confidence interval between 0.29 and 0.37 mg/L).
Executive summary:

A full OECDTG 202 GLP test was performed with Daphnia magna, based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Copaiba Balsam oil were prepared and used as test concentrations. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared at loading rates of 0.46 and 1.0 mg/L under static conditions. In addition, solutions containing 4.6, 10, 22 and 46% of the lower WAF were prepared and used as lower test concentrations.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. The exposure was performed in airtight closed vessels with headspace reduced to minimum. The test item appeared to be poorly soluble in test medium. Despite the fact that no clear dose-related increase of concentration could be observed, immobilisation increased with the applied dose. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EL50 for Daphnia magna exposed to Copaiba balsam oil was 0.33 mg/L (95% confidence interval between 0.29 and 0.37 mg/L).

Description of key information

A full OECDTG 202 GLP test was performed with Daphnia magna, based on the results of a preceding range-finding test. Water Accommodated Fractions (WAFs) of Copaiba Balsam oil were prepared and used as test concentrations. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared at loading rates of 0.46 and 1.0 mg/L under static conditions. In addition, solutions containing 4.6, 10, 22 and 46% of the lower WAF were prepared and used as lower test concentrations.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. The exposure was performed in airtight closed vessels with headspace reduced to minimum. The test item appeared to be poorly soluble in test medium. Despite the fact that no clear dose-related increase of concentration could be observed, immobilisation increased with the applied dose. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EL50 for Daphnia magna exposed to Copaiba balsam oil was 0.33 mg/L (95% confidence interval between 0.29 and 0.37 mg/L).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.33 mg/L

Additional information