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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-Nov-16 to 13-Jan-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13-Apr-2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
May 2008
Deviations:
no
Principles of method if other than guideline:
- Principle of test: The test was modified according to the OECD Series on Testing and Assessment Document no. 23 on aquatic toxicity testing of difficult substances (Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000).
- Short description of test conditions: water accomated fractions were prepared to create a series of loading rates to be tested.
- Parameters analysed / observed: See below.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Every tested concentration and the control
- Sampling method: For sampling 4.0 mL from the approximate centre of the test vessels was taken at the start and end of each 24 hour renewal period over 48 hours (i.e. at 0 hours freshly dosed medium was analysed, 24 hours old medium and freshly dosed medium was analysed and at 48 hours old medium was analysed only). At the end of each exposure period the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: The samples were stored in a freezer (<-15 °C) until analysis stability at this storge temperature as been previously confirmed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Test medium used was Adjusted ISO medium.

- Method: The main test was conducted at 1.0, 1.8, 3.2, 5.6 10 % of a 100 mg/L saturated solution. Test solutions were prepared from a stock of saturate solution. The solution was obtained by stirring 100 mg/L of test item in the test medium for 2 days. This was followed by a 1 hour settling period. After this time the soluble fraction was siphoned from the vessel for use as the test medium.
- Eluate: N/A
- Differential loading: N/A
- Controls: A control consisted of the test medium only.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): analytic, gas chomatograph of main constituents.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: N/A (In-house culture;
- Justification for species other than prescribed by test guideline: N/A (guideline species)
- Source: In-house laboratory culture with a known history (Stocks were kept in Elendt M7 medium and fed daily with fresh water algae. After 7 days of cultivation half of the medium twice a week, cultures were not allowed to exceed an age of 4 weeks).
- Age of parental stock (mean and range, SD): 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. Parental daphnids were more than two weeks old.
- Feeding during test: None
- Food type: N/A
- Amount: N/A
- Frequency: N/A

ACCLIMATION
- Acclimation period: N/A
- Acclimation conditions (same as test or not): ISO medium is used instead of Elendt M7 medium
- Type and amount of food: N/A
- Feeding frequency: N/A
- Health during acclimation (any mortality observed): N/A

QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
Consistent with guideline requirement.
Post exposure observation period:
Not applicable
Hardness:
180 mg/L CaCO3
Test temperature:
20 to 21°C
pH:
7.8 to 8.0
Dissolved oxygen:
8.9 to 9.6 mg/L
Salinity:
N/A
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal concentrations were based on a percentage of a saturated solution. The saturated solution was obtained by dissolving 100 mg/L of test item in test medium for 48 hours, whilst being stirred continuously. After 48 hours the vessel was allowed to settle for 1 hour. At this point the soluble fraction was removed. Using this Saturated solution (and ensuring no solids were present by using the Tyndalls effect) concentrations of 1.0, 1.8, 3.2, 5.6 and 10 % were made.

Analysis of the samples taken at the start of the first renewal period showed measured concentrations of 0.76, 1.5, 2.5, 4.2 and 7.6 mg/L at 1.0, 1.8, 3.2, 5.6 and 10% of the SS at 100 mg/L, respectively. All concentrations remained stable during the first renewal period (93-100% of initial at the end of the renewal period). At the start of the second renewal period measured concentrations were 0.88, 1.6, 2.8, 4.7 and 8.4 mg/L at 1.0, 1.8, 3.2, 5.6 and 10% of the SS at 100 mg/L, respectively. All concentrations remained stable during the second renewal period (86-92% of initial at the end of the renewal period). Therefore, the effect parameters were based on the average of the initial measured concentrations that were calculated to be 0.82, 1.5, 2.7, 4.4 and 8.0 mg/L.
Details on test conditions:
Firstly a combined limit/range finder test was conducted in order to determine if 1) a definitive test was required, and/or 2) what concntration range should be used in the definitive test. Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a control and a saturated solution at 100 mg/L at 100 %. The test was performed under static conditions (i.e. no renewal of test solutions), ten daphnids per concentration (two replicates, 5 daphnids per replicate) were also exposed to 1.0 and 10 % of the saturated solution. Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration. The test was conducted in Adjusted ISO medium.

Test medium used was Adjusted ISO medium. The main test was conducted at 1.0, 1.8, 3.2, 5.6 10 % of a 100 mg/L saturated solution. A control consisted of the test medium only. At each concentration 20 daphnids were exposed split into 4 replicates of 5 dapnids each in 80 mL medium/ medium+test item. The test was conducted for 48 hours and renewel occured every 24 hours. Daphnids were not fed during the test, there was no aeration. The daphnids were introduced within 40 minutes of the test meidum preperation. Immobility was measured at 24 and 48 hours. Dissolved oxygen and pH was measured at the start and end of each renewal period, temperature was continuously monitored. The Spearman-Karber test was used to estimate effect levels due to deviations in monotonicity of the dataset. No trim was applied to reflect a worst-case EC50 value.

The test was considered valid if:

- Control daphnid immobility did not exceed 10 %, and no signs of disease or stress were displayed (i.e. surface trapping, unusual behaviour and discolouration).
- Dissolved oxygne within the medium stayed at >= 3 mg/L in all vessels.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: In the control one daphnid was found surface trapped in one replicate. In one replicate for both 10 and 100 mg/L of test material in one replicate, 4 and 2 daphnids were found to be surface trapped respectively.
- Observations on body length and weight: N/A
- Other biological observations: N/A
- Mortality of control: No mortality
- Other adverse effects control: surface entrapment of one daphnid.
- Abnormal responses: surface entrapment in 2 of the upper connctrartions.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.

For the range finder/limit test the accuracy target fell outwith the concentration criterion for 0.1 mg/L (i.e. 67 %). However, for 100 mg/L and for the definitive test the recovery/accuracy of the test item measurements were within the criterion (70-110%) and ranged from 82-93 % and therefore did not effect the validity of the final definitive test. During the definitive test the test medium measurements were of an acceptable level and fell within the criterion of 70-110 %.
No undissolved particles were observed in the test solution stock (using the Tyndall effect). Validity criteria for the test were met.

Measured concentrations in the definitive test were 0.76, 1.5, 2.5, 4.2 and 7.6 mg/L at 1.0, 1.8, 3.2, 5.6 and 10% of the saturated solution at 100 mg/L, respectively. All concentrations remained stable during the first renewal period (93-100% of initial at the end of the renewal period). At the start of the second renewal period measured concentrations were 0.88, 1.6, 2.8, 4.7 and 8.4 mg/L at 1.0, 1.8, 3.2, 5.6 and 10% of the SS at 100 mg/L, respectively. All concentrations remained stable during the second renewal period (86-92% of initial at the end of the renewal period). Therefore, the effect parameters were based on the average of the initial measured concentrations that were calculated to be 0.82, 1.5, 2.7, 4.4 and 8.0 mg/L.

All daphnids exposed to 8.0 mg/L became immobilized within 24 hours of exposure. In addition, 55% of the daphnids exposed to 4.4 mg/L became immobilized, while no immobility was observed at any of the lower concentrations and the control.

After 48 hours of exposure immobility was observed at all test concentrations. In the concentrations up to and including 2.7 mg/L immobility was below 50%, while immobility in concentrations of 4.4 mg/L and higher was above 50%.

For full results see tables in results section.
Results with reference substance (positive control):
- Results with reference substance valid? yes, potassium dichromate was used and the test with the validity criteria as set within the guideline.
- Relevant effect levels: EC50 at 24 and 48 hours
- Limit test: no
- Dose-response test: yes
- ECx: The 24h-EC50 was 4.4 mg/L with a 95% confidence interval ranging from 3.9 to 5.0 mg/L. The 48h-EC50 was 2.2 mg/L with a 95% confidence interval ranging from 1.8 to 2.6 mg/L.
- Other: N/A
Reported statistics and error estimates:
The 24h-EC50 was 4.4 mg/L (95% confidence interval between 3.9-5.0 mg/L).
The 48h-EC50 was 2.7 mg/L (95% confidence interval between 1.8-2.6 mg/L).

Number of introduced daphnids and incidence of immobility in the final test

Time (h)

Replicate

Average initial concentration Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal (mg/L)

Control

0.82

1.5

2.7

4.4

8.0

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0 [2]

0

0

1

5

B

0

0

0

0

4

5

C

0

0

0

0

3

5

D

0

0

0

0

3

5 [2] 

Total immobilised

0

0

0

0

11

20

Effect %

0

0

0

0

55

100

 

 

 

 

 

 

48

A

0

1

1

3

5

5

B

0

2

0

3

5

5

C

0

1

1 [1] 

2

5

5

D

0

1

0

1

5

5

Total immobilised

0

5

2

9

20

20

Effect %

0

25

10

45

100

100

[ ]: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

pH and oxygen concentrations (mg/L) during the final test

Average initial conc. Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal

(mg/L)

Start (t=0 h)

t=24h old

t=24h fresh

End (t=48 h)

 

pH

 

O2

 

pH

 

O2

 

pH

 

O2

 

pH

 

O2

control

8.0

9.2

7.8

8.9

7.8

9.3

7.9

9.6

0.82

8.0

9.1

7.9

8.9

7.8

9.2

7.9

9.6

1.5

8.0

9.1

7.9

8.9

7.8

9.1

7.9

9.6

2.7

8.0

9.0

7.9

8.9

7.9

9.1

7.9

9.6

4.4

7.9

9.0

7.9

8.9

7.9

9.1

7.9

9.6

8.0

7.9

9.0

7.9

8.9

7.8

9.1

7.9

9.6

Concentrations of the test item in test medium - final test

Time of sampling
[hours]

Date of sampling

Date of
analysis

Percentage of Saturated solution
[%]

Analysed concentration
[mg/L]

Relative to
initial
[%]

 

 

 

 

 

 

0

02 Jan 2017

10 Jan 2017

0

n.d.

 

 

 

 

1.0

0.764

 

 

 

 

1.8

1.49

 

 

 

 

3.2

2.52

 

 

 

 

5.6

4.16

 

 

 

 

10

7.63

 

 

 

 

 

 

 

24

03 Jan 2017

10 Jan 2017

0

n.d.

n.a.

(old)

 

 

1.0

0.740

97

 

 

 

1.8

1.47

99

 

 

 

3.2

2.35

93

 

 

 

5.6

4.13

99

 

 

 

10

7.59

100

 

 

 

 

 

 

24

03 Jan 2017

10 Jan 2017

0

n.d.

 

(fresh)

 

 

1.0

0.882

 

 

 

 

1.8

1.58

 

 

 

 

3.2

2.80

 

 

 

 

5.6

4.69

 

 

 

 

10

8.43

 

 

 

 

 

 

 

48

04 Jan 2017

10 Jan 2017

0

n.d.

n.a.

(old)

 

 

1.0

0.756

86

 

 

 

1.8

1.39

88

 

 

 

3.2

2.45

87

 

 

 

5.6

4.14

88

 

 

 

10

7.76

92

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
The 48h-EC50 of the test item to Daphnia magna was 2.2 mg/L (95% confidence interval between 1.8-2.6 mg/L).
Executive summary:

Acute Toxicity Study in Daphnia magnawith Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the CommissionRegulation (EC) No 440/2008, Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

The batch of Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal tested was a colourless liquid with a purity of 99.6% (sum of 2 diastereomers). The test item was not completely soluble in test medium at the loading rate initially prepared.

A semi-static final test was performed based on the results of a preceding static combined limit/range-finding test. An initial stock solution at 100 mg/L was prepared daily by adding measured amounts of the test item to test medium and mixing for 2 days to achieve an equilibrated concentration of dissolved constituents in the aqueous phase. Following cessation of mixing, and a 1 hour period of settling, the saturated solution (SS) was siphoned out and checked for tyndall effect, using a Laser pen. The clear and colourless SS was used as a stock solution. Lower concentrations were obtainedviaserial dilution of the SS in test medium.

Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to 1.0, 1.8, 3.2, 5.6 and 10% of the SS prepared at 100 mg/L. The total exposure period was 48 hours and test solutions were renewed after 24 hours of exposure.Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of both renewal periods.

Analysis of the samples taken at the start of the first renewal period showed measured concentrations of 0.76, 1.5, 2.5, 4.2 and 7.6 mg/L at 1.0, 1.8, 3.2, 5.6 and 10% of the SS at 100 mg/L, respectively. All concentrations remained stable during the first renewal period (93-100% of initial at the end of the renewal period). At the start of the second renewal period measured concentrations were 0.88, 1.6, 2.8, 4.7 and 8.4 mg/L at 1.0, 1.8, 3.2, 5.6 and 10% of the SS at 100 mg/L, respectively. All concentrations remained stable during the second renewal period (86-92% of initial at the end of the renewal period). Therefore, the effect parameters were based on the average of the initial measured concentrations that were calculated to be 0.82, 1.5, 2.7, 4.4 and 8.0 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 48h-EC50was 2.2 mg/L (95% confidence interval between 1.8 and 2.6 mg/L).

Description of key information

48 h EC50 (Daphnia magna) = 2.2 mg/L, OECD 202 Daphnia sp. acute immobilisation test, (Anon. 2017)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.2 mg/L

Additional information

Acute Toxicity Study in Daphnia magnawith Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No 440/2008, Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

The batch of Reaction mass of (3R)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal and (3S)-3-[(1R)-4-Methyl-3-cyclohexen-1-yl]butanal tested was a colourless liquid with a purity of 99.6% (sum of 2 diastereomers). The test item was not completely soluble in test medium at the loading rate initially prepared.

A semi-static final test was performed based on the results of a preceding static combined limit/range-finding test. An initial stock solution at 100 mg/L was prepared daily by adding measured amounts of the test item to test medium and mixing for 2 days to achieve an equilibrated concentration of dissolved constituents in the aqueous phase. Following cessation of mixing, and a 1 hour period of settling, the saturated solution (SS) was siphoned out and checked for tyndall effect, using a Laser pen. The clear and colourless SS was used as a stock solution. Lower concentrations were obtainedviaserial dilution of the SS in test medium.

Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to a control and to 1.0, 1.8, 3.2, 5.6 and 10% of the SS prepared at 100 mg/L. The total exposure period was 48 hours and test solutions were renewed after 24 hours of exposure.Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of both renewal periods.

Analysis of the samples taken at the start of the first renewal period showed measured concentrations of 0.76, 1.5, 2.5, 4.2 and 7.6 mg/L at 1.0, 1.8, 3.2, 5.6 and 10% of the SS at 100 mg/L, respectively. All concentrations remained stable during the first renewal period (93-100% of initial at the end of the renewal period). At the start of the second renewal period measured concentrations were 0.88, 1.6, 2.8, 4.7 and 8.4 mg/L at 1.0, 1.8, 3.2, 5.6 and 10% of the SS at 100 mg/L, respectively. All concentrations remained stable during the second renewal period (86-92% of initial at the end of the renewal period). Therefore, the effect parameters were based on the average of the initial measured concentrations that were calculated to be 0.82, 1.5, 2.7, 4.4 and 8.0 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 48h-EC50was 2.2 mg/L (95% confidence interval between 1.8 and 2.6 mg/L).