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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 January 2010 to 25 February 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
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:
Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 ºC for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION- Method: 160 mg test substance was dissolved directly in culture medium and the volume adjusted to 1 litre to give a 160 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 80, 40, 20 and 10 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (11.8 mL) to give the required test concentrations of 10, 20, 40, 80 and 160 mg/L. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM- Strain: CCAP 276/20- Source: Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, UK - Culture maintenance: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 ºC.ACCLIMATION- Culturing media and conditions: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10³ cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 ºC until the algal cell density was approximately 10^4 to 10^5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 ºC
pH:
7.1 - 7.6
Nominal and measured concentrations:
Control, 10, 20, 40, 80, 160 mg/L (nominal)< LOQ, 1.0, 7.1, 23, 52, 113 mg/L (geometric mean measured test concenttrations)
Details on test conditions:
TEST SYSTEM- Test vessel: 250 mL glass conical flasks - Type: closed (conical flasks were plugged with polyurethane bungs)- Fill volume: 100 mL- Initial cell density: 4 x 10³ cells/mL- No. of vessels per concentration: triplicate- No. of vessels per control: sixGROWTH MEDIUM- Standard medium used: yes- The culture medium used was the same as that used to maintain the stock culture The culture medium was composed of the following: NaNO3 (25.5 mg/L), MgCl2.6H2O (12.164 mg/L), CaCl2.2H2O (4.41 mg/L), MgSO4.7H2O (14.7 mg/L), K2HPO4 (1.044 mg/L), NaHCO3 (15 mg/L), H3BO3 (0.1855 mg/L), MnCl2.4H2O (0.415 mg/L), ZnCl2 (0.00327 mg/L), FeCl3.6H2O (0.159 mg/L), CoCl2.6H2O (0.00143 mg/L), Na2MoO4.2H2O (0.00726 mg/L), CuCl2.2H2O (0.00012 mg/L), Na2EDTA.2H2O (0.3 mg/L), Na2SeO3.5H2O (0.00001 mg/L)TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)- Adjustment of pH: with 0.1N NaOH or HCl to pH 7.5 ± 0.1OTHER TEST CONDITIONS- Photoperiod: continuous illumination- Light intensity and quality: approximately 7000 lux provided by warm white lighting (380 - 730 nm)EFFECT PARAMETERS MEASURED :Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle CounterRANGE FINDING STUDY- Test concentrations: 0.10, 1.0, 10 and 100 mg/L (in duplicate)- Test conditions: as for definitive study- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
110 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
52 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
52 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Growth rate (r) and yield (y) of Desmodesmus subspicatus were affected by the presence of the test substance over the 72 hour exposure period. Refer to results tables in "Any other information on results incl. tables" for further information.

The data is being used in a read-across approach to address toxicity to aquatic algae and cyanobacteria of the registered substance, 1-phenylethyl acetate (see target record). The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus the 72-h EC50 and NOEC based on growth rate have been chosen as the key values for read-across and classification purposes.

Range-finding Test

No effects on growth were seen at test concentrations of 0.1, 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L. Based in this information, test concentrations of 10, 20, 40, 80 and 160 mg/L were selected for the definitive test.

Definitive Test

Growth rate (r) and yield (y) of Desmodesmus subspicatus were affected by the presence of the test material over the 72 hour exposure period.

Table 1: Mean Cell Densities and pH Values - Definitive Test

Nominal conc (mg/L)

pH

Cell densities (cells/mL) mean values at time

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

7.1

4.30 x 10^3

1.11 x 10^4

2.36 x 10^4

1.02 x 10^5

7.6

10

7.1

4.21 x 10^3

1.07 x 10^4

2.47 x 10^4

9.79 x 10^4

7.5

20

7.1

4.54 x 10^3

9.26 x 10^3

2.19 x 10^4

1.14 x 10^5

7.5

40

7.1

4.40 x 10^3

6.70 x 10^3

2.09 x 10^4

1.03 x 10^5

7.5

80

7.1

4.29 x 10^3

7.58 x 10^3

2.30 x 10^4

1.37 x 10^5

7.5

160

7.1

4.40 x 10^3

4.68 x 10^3

1.03 x 10^4

2.42 x 10^4

7.3

 

Table 2: Inhibition of Growth Rate and Yield - Mean Values in the Definitive Test

Nominal conc (mg/L)

Growth rate (cells/mL/hour)

Yield (cells/mL)

0 - 72 h

% inhibition

0 - 72 h

% inhibition

Control

0.045

9.76 x 10^4

10

0.045

1

9.37 x 10^4

4

20

0.047

[3]

1.10 x 10^5

[12]

40

0.045

[1]

9.86 x 10^4

[1]

80

0.049

[9]

1.33 x 10^5

[36]

160

0.025

44

1.98 x 10^4

80

[increase in growth as compared to controls]

Inhibition of growth rate

ErC10 (0 - 72 h) = 150 mg/L

ErC20 (0 - 72 h) = 150 mg/L

ErC50 (0 - 72 h) = 160 mg/L

Inhibition of yield

EyC10 (0 - 72 h) = 100 mg/L

EyC20 (0 - 72 h) = 110 mg/L

EyC50 (0 - 72 h) = 130 mg/L

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 10, 20, 40 and 80 mg/L, however, no intact cells were observed to be present in the test culture at 160 mg/L.

Observations on Test Substance Solubility

At the start of the test all control, 10, 20, 40, 80 and 160 mg/L test cultures were observed to be clear colourless solutions. After the 72 hour test period all 10, 20, 40 and 80 mg/L test cultures were observed to be pale green dispersions whilst the 160 mg/L test cultures were observed to be clear colourless solutions.

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal. A concentration dependent decline in measured concentrations was observed at 72 hours in the range of 1% to 50% of nominal. This decline indicated that the test substance was unstable, particularly at the lower test concentrations employed. A further decline in measured concentrations was considered to be due to adsorption of the test substance to the algal cells present. Adsorption was not a factor in the preliminary stability analyses conducted as no algal cells were present. Results are therefore based on the geometric mean measured test concentrations in order to give a 'worst case' analysis of the data. The geometric mean measured test concentrations were determined to be:

Nominal test conc

(mg/L)

Geometric mean measured test conc (mg/L)

Expressed as a % of nominal

10

1.0

10

20

7.1

36

40

23

58

80

52

65

160

113

71

The following results were determined from the data based on the geometric mean measured test concentrations:

Growth rate

ErC10 (0 - 72 h) = 100 mg/L

ErC20 (0 - 72 h) = 110 mg/L

ErC50 (0 - 72 h) = 110 mg/L

Yield

EyC10 (0 - 72 h) = 67 mg/L

EyC20 (0 - 72 h) = 75 mg/L

EyC50 (0 - 72 h) = 92 mg/L

NOEC = 52 mg/L

LOEC = 113 mg/L

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, benzyl acetate was determined to have 72h EC50 values, based on geometric means, of 110 mg/L (growth rate) and 92 mg/L (yield). The NOEC was determined to be 52 mg/L (growth rate and yield) and the LOEC to be 113 mg/L (growth rate and yield).

The data is being used in a read-across approach to address the toxicity to aqautic algae and cyanobacteria of the registered substance, 1-phenylethyl acetate (see target record). The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus the 72-h EC50 and NOEC based on growth rate have been chosen as the key values for read-across and classification purposes.
Executive summary:

The effect of the test substance on the growth of the green alga Desmodesmus subspicatus was determined according to standardised guidelines OECD 201 and EU Method C.3. Following a preliminary range-finding test, D. subspicatus was exposed to an aqueous solution of the test substance at concentrations of 10, 20, 40, 80 and 160 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 ºC. Samples of the algal population were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal. A concentration dependent decline in measured concentrations was observed at 72 hours in the range of 1% to 50% of nominal. This decline indicated that the test substance was unstable, particularly at the lower test concentrations employed. A further decline in measured concentrations was considered to be due to adsorption of the test substance to the algal cells present. Adsorption was not a factor in the preliminary stability analyses conducted as no algal cells were present. Results are therefore based on the geometric mean measured test concentrations in order to give a 'worst case' analysis of the data.

Exposure of D. subspicatus to the test substance gave the following results based on the geometric measured test concentrations:

Response variable

EC50 (mg/L)

NOEC (mg/L)

LOEC (mg/L)

Growth rate

110

52

113

Yield

92

52

113

Description of key information

The toxicity to aquatic algae and cyanobacteria 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 110 mg/L according to a study performed in line with OECD Guideline 201 and EU Method C.3.

Key value for chemical safety assessment

EC50 for freshwater algae:
110 mg/L
EC10 or NOEC for freshwater algae:
52 mg/L

Additional information

Reliable measured toxicity data to aquatic algae and cyanobacteria is not available for 1-phenylethyl acetate. Since a valid study is available for the analogue substance, benzyl acetate, a read-across approach has been used. The effect of the test substance on the growth of the green alga Desmodesmus subspicatus was determined according to standardised guidelines OECD 201 and EU Method C.3. Following a preliminary range-finding test, D. subspicatus was exposed to an aqueous solution of the test substance at concentrations of 10, 20, 40, 80 and 160 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 ºC. Samples of the algal population were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal. A concentration dependent decline in measured concentrations was observed at 72 hours in the range of 1% to 50% of nominal. This decline indicated that the test substance was unstable, particularly at the lower test concentrations employed. A further decline in measured concentrations was considered to be due to adsorption of the test substance to the algal cells present. Adsorption was not a factor in the preliminary stability analyses conducted as no algal cells were present. Results are therefore based on the geometric mean measured test concentrations in order to give a 'worst case' analysis of the data.

Exposure of D. subspicatus to the test substance gave the following results based on the geometric measured test concentrations:

Response variable

EC50 (mg/L)

NOEC (mg/L)

LOEC (mg/L)

Growth rate

110

52

113

Yield

92

52

113

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.

The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). Thus the ErC50 is used for classification purposes.