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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 February 2011 to 18 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Received from International Flavors & Fragrances, a liquid, identified as 00022255 BENZ ACET PRG benzyl acetate; Lot Number: SM10033061
- Expiration date of the lot/batch: March 2011
- Purity test date: 99.9%
Analytical monitoring:
yes
Details on sampling:
Samples were collected from one test chamber of each treatment and control group one day prior to the start of the test after conditioning the diluter for 24 hours. Samples were also collected from alternating replicate chambers in each treatment and control group at the beginning of the test and at 48 hours. The samples were collected from mid-depth, placed in glass vials, and processed immediately for analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION : Individual stock solutions were prepared for each of the five concentrations tested. A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 600 mg/mL. Four secondary stock solutions at nominal concentrations of 75, 150, 300 and 450 mg/L were prepared in DMF by proportional dilution of the primary stock. The stock solutions were mixed by inversion and were clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM- Source: Wildlife International, Ltd., Easton, Maryland, USA- Age at study initiation: <24 hours- Method of breeding: Five adult daphnids were were used to supply neonates for the test were held for at least 22 days prior to collection of the juveniles for testing, and had each produced at least one previous brood- Feeding during test: Neonates were not fed during the test. Adult daphnids were fed daily a mixture of yeast, cereal grass media and trout chow, as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitataACCLIMATION (adult daphnids)- Acclimation period: 14 days- Acclimation conditions: same as test - Health during acclimation (any mortality observed): The adults showed no signs of disease or stress
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
140 mg/L
Test temperature:
20 ± 1 ºC
pH:
8.0 - 8.1
Dissolved oxygen:
≥ 7.5 mg/L (≥ 83 % of saturation)
Nominal and measured concentrations:
0, 7.5, 15, 30, 45, 60 mg/L (nominal)
< LOQ, 6.5, 13, 26, 39, 51 mg/L (measured)
Details on test conditions:
TEST SYSTEM- Test vessel: 25 L stainless steel aquaria - Fill volume of test vessels: 22 L- Type of flow-through: proportional diluter- Renewal rate of test solution (frequency/flow rate): 5 volume additions of test water in each test chamber per day- No. of organisms per vessel: 10 - No. of vessels per concentration: tests were conducted in duplicate- No. of vessels per control: tests were conducted in duplicate- No. of vessels per vehicle control: tests were conducted in duplicateTEST MEDIUM / WATER PARAMETERS- Source of dilution water: well water (40 m deep located on the Wildlife International, Ltd., site). The water was passed through a sand filter to remove particles greater than 25 µm before being aerated. Prior to use, the water was filtered through a 0.45 µm filter and passed through a UV steriliser.- Alkalinity: 174 mg/L- pH: 8.2- Conductivity: 358 µS/cm- Hardness: 138 mg/L- Culture medium different from test medium: noOTHER TEST CONDITIONS- Photoperiod: 16 hours light / 8 hours dark with a 30 minute transition periodEFFECT PARAMETERS MEASURED (with observation intervals if applicable) : All organisms were observed for immobility, signs of toxicity or abnormal behaviour at 1.5, 24 and 48 hours after test initiation
Reference substance (positive control):
not specified
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 51 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
37 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.I. 31.5-44.3 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
< 6.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Details on results:
With the exception of one daphnid in the negative control noted as floating on the surface at the 48-hour interval and four daphnids in the solvent control that were floating at the 24-hour interval, daphnia in the negative and control groups appeared normal throughout the test, with no immobility or signs of toxicity. Percent immobility at test termination in the 6.5, 13, 26, 39 and 51 mg/L treatment groups was 0, 0, 20, 70 and 65% respectively. Floating daphnids were also observed in all treatment groups at the initial observation approximately 1.5 hours after test initiation, and at the 24 and 48 hour observations. All floating daphnids in the controls and treatment groups appeared normal after being gently submerged. Lethargy was also observed for one or two daphnids each in the 26, 39 and 51 mg/L treatment groups.

The data is being used in a read-across approach to address the short-term toxicity to daphnia of the registered substance, 1-phenylethyl acetate (see target record).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, benzyl acetate was determined to have a 48 hour EC50 to Daphnia magna in a flow-through test system, based on mean measured concentrations, of 37 mg/L.

The data is being used in a read-across approach to address the short-term toxicity to daphnia of the registered substance, 1-phenylethyl acetate (see target record).
Executive summary:

The EC50 for immobilsation of Daphnia exposed to the test substance, benzyl acetate, was determined over a 48 hour period in a study conducted in line with standardised guidelines OECD 202 and EPA OPPTS 850.1010.

The nominal concentrations of test substance were 0, 7.5, 15, 30, 45 and 60 mg/L with the corresponding overall mean measured concentrations being 0, 6.5, 13, 26, 39 and 51 mg/L, respectively. Chemical analysis of samples taken at 0 and 48 hours indicate that the concentrations were reasonably well maintained over the test period. The test substance was added to the test system with DMF as a co-solvent. No undissolved test substance was observed in the test vessels. Two vessels were prepared at each test concentration with a solvent and non-solvent control included. Ten Daphnia were added to each test vessel.

Under the conditions of the test, the 48 hour EC50 to Daphnia magna in a flow-through test system, based on mean measured concentrations, was 37 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25 January 2010 to 12 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name: Benzyl Acetate
Description: clear colorless liquid
Batch Number: 75467
Date Received: 21 Dec 2009
Expiry date: 21 June 2010
Purity: 99.89% (benzyl alcohol present at 0.014%)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control, 1.0, 3.2, 10, 32 and 100 mg/L test groups (replicates R1 - R2 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) for immediate quantitative analysis. Duplicate samples were taken and stored at approximately -20 ºC for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: 200 mg test substance was dissolved in reconstituted water and the volume adjusted to 2 litres to give the 100 mg/L test concentration from which serial dilutions were made to give further test concentrations of 56, 32, 18, 10, 5.6, 3.2, 1.8 and 1.0 mg/L. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM- Source: in-house laboratory cultures - Age at study initiation: 24 hours- Method of breeding: gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were approximately 24 hours old at test initiation- Feeding: adult daphnids were fed daily with a suspension of algae (Chlorella sp.). Daphnids received no food during exposure.ACCLIMATION- Acclimation conditions: same as test
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
19 - 20 ºC
pH:
7.7 - 8.0
Dissolved oxygen:
8.9 - 9.7 mg O2/L
Nominal and measured concentrations:
Control, 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56, 100 mg/L (nominal)< LOQ, 0.977, -, 3.03, -, 10.3, -, 33.7, -, 106 mg/L (measured at 0 hours)< LOQ, 0.806, -, 2.71, -, 9.64, -, 31.0, -, 96.0 mg/L (measured at 24 hours - old media)< LOQ, 0.978, -, 3.25, -, 10.4, -, 32.4, -, 103 mg/L (measured at 24 hours - fresh media)< LOQ, 0.861, -, 2.93, -, 10.0, -, 32.6, -, 105 mg/L (measured at 48 hours - old media)
Details on test conditions:
TEST SYSTEM- Test vessel: 250 mL glass jars- Type (delete if not applicable): closed- Fill volume: 200 mL- Aeration: no- Renewal rate of test solution: test media renewal at 24 hours (test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-hour old test media into the fresh test media)- No. of organisms per vessel: 10- No. of vessels per concentration: duplicate- No. of vessels per control: duplicateTEST MEDIUM / WATER PARAMETERS- Dilution water: reconstituted water- Preparation: 25 mL of each of CaCl2.2H2O (11.76 g/L), MgSO4.7H2O (4.93 g/L), NaHCO3 (2.59 g/L) and KCl (0.23 g/L) was added to 1L deionised water.- pH: 7.8 ± 0.2 (adjusted if necessary with NaOH or HCl)- Hardness: 250 mg/L as CaCO3- The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation valueOTHER TEST CONDITIONS- Photoperiod: 16 hours light / 8 hours dark with 20 minute dawn and dusk transition periodsEFFECT PARAMETERS MEASURED: Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of the exposure. the criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.RANGE FINDING STUDY- Test concentrations: 0, 0.10, 1.0, 10 and 100 mg/L- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.I. 20 - 30 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.I. 14 - 20 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: No significant immobilisation were seen at this concentrtaion
Details on results:
Immobilisation data from exposure of Daphnia magna to the test substance are presented in Table 2 in the field "Any other information on results incl. tables".

The data is being used in a read-across approach to address the short-term toxicity to daphnia of the registered substance, 1-phenylethyl acetate (see target record).
Results with reference substance (positive control):
Immobilisation data from exposure of Daphnia magna to the reference substance are presented in Table 3 in the field "Any other information on results incl. tables". Results were within the normal range expected.

Verification of test concentrations

Analysis of the test preparations at 0, 24 and 48 hours showed measured test concentrations to range from 8% to 106% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Range-Finding Test

Cumulative immobilisation data are as follows:

Table 1: Cumulative Immobilisation Data in the Range-Finding Test

Nominal conc. (mg/L)

Cumulative immobilised Daphnia (initial population: 10/replicate)

24 hours

48 hours

Control

0

0

0.1

0

0

1.0

0

0

10

0

3

100

9

10

 

Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Definitive test

Table 2: Cumulative Immobilisation Data in the Definitive Test

Nominal conc. (mg/L)

Cumulative immobilised Daphnia (initial population: 10/replicate)

24 hours

48 hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

1.0

0

0

0

0

0

0

0

0

1.8

0

0

0

0

0

0

0

0

3.2

0

0

0

0

0

0

0

0

5.6

0

1

1

5*

0

1

1

5*

10

0

0

0

0

0

0

0

0

18

3

2

5

25

6

5

11

55

32

7

9

16

80

9

10

19

95

56

8

9

17

85

10

10

20

100

100

10

10

20

100

10

10

20

100

R1, R2 = Replicates 1 and 2

* single immobilised daphnid considered to be due to natural causes rather than true toxicity

Table 3: Cumulative Immobilisation Data in the Positive Control

Nominal conc. (mg/L)

Cumulative immobilised Daphnia (initial population: 10/replicate)

24 hours

48 hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

0.32

0

0

0

0

0

0

0

0

0.56

1

1

2

10

3

2

5

25

1.0

7

7

14

70

10

10

20

100

1.8

10

10

20

100

10

10

20

100

3.2

10

10

20

100

10

10

20

100

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, benzyl acetate was determined to have a 48-hour EC50 of 17 mg/L. The No Observed Effect Concentration at 48 hours was determined to be 10 mg/L.

The data is being used in a read-across approach to address the short-term toxicity to daphnia of the registered substance, 1-phenylethyl acetate (see target record).
Executive summary:

The acute toxicity of the test substance to Daphnia magna was determined in line with standardised guidelines OECD 202 and EU Method C.2. Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test substance at concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of approximately 20ºC under semi static conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

Under the conditions of the study, the 48 hour EC50 for the test substance to D. magna, based on nominal test concentrations was 17 mg/L with 95% confidence limits of 14-20 mg/L. The NOEC was determined to be 10 mg/L. Analysis of the test preparations at 0, 24 and 48 hours showed measured test concentrations to range from 81% to 106% of nominal value and so the results were based on the nominal concentrations only.

Description of key information

The short-term toxicity to aquatic invertebrates of 1-phenylethyl acetate has been assessed using a read-across approach from the analogue substance benzyl acetate. The EC50 of benzyl acetate was determined to be 25 mg/L according the geometric mean of two studies (Gallagher et al., 2011 - 17 mg/L; Vryenhoef and Mullee, 2010 – 37 mg/L), the geometric mean selected in line with 'Guidance on information requirements and chemical safety assessment' Chapter R.7b. Both studies were performed in line with appropriate standardised guidelines and to GLP.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
25 mg/L

Additional information

Reliable measured short-term toxicity data to aquatic invertebrates is not available for 1-phenylethyl acetate. Since two valid studies are available for the analogue substance benzyl acetate, a read-across approach has been used. The two available studies determined the acute toxicity of benzyl acetate to Daphnia magna to be EC50 37 mg/L and 17 mg/L, respectively. Since the two studies are of the same reliability, and the EC50 values do not vary by more than one order of magnitude, the geometric mean of the EC50 values has been determined in accordance with ECHA guidance chapter R.7b. The read-across is considered to be suitable based on the structural and 'mechanistic action' similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.