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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 to 25 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control (replicate 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 °C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
The test item was dissolved directly in culture medium.
An amount of test item (160 mg) was dissolved 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 concentration of 10, 20, 40, 80 and 160 mg/L.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogenity.
The concentration and stability of the test item in the test preparations were verified by chemicalc analysis at 0 and 72 hours.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation):
- Method of cultivation: Master cultures were maintained i nthe laboratory by the periodic replenishment of culture mdeium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

ACCLIMATION
Prior to the start of the test sufficent master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 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 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperatures were maintained at 24 ± 1°C for the duration of the study.
pH:
0 hours: pH 7.1
72 hours: pH 7.6
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal exposure concentrations; control, 10, 20, 40, 80 and 160 mg/L.
Geometric mean measured concentrations - 1.0, 7.1, 23, 52 and 113 mg/L.
Details on test conditions:
250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.70 x 10 ^5 cells per mL. Inoculation of 500 mL of test medium with 11.8 mL of this algal suspension gave an initial nominal cell density of 4 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
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:
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:
LOEC
Effect conc.:
113 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
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
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
113 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Growth Data:
It is clear from the results that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-hour exposure period.

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 cultures at 160 mg/L.

Observations on Test Item 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.

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

Growth rate:
ErC10 (0 - 72 h): 110 mg/L
ErC20 (0 - 72 h): 110 mg/L
ErC50 (0 - 72 h): 110 mg/L
NOEC = 52 mg/L
LOEC = 113 mg/L

Inhibition of 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
Results with reference substance (positive control):
The results for Potassium Dichromate were within the normal ranges for this reference item.

ErC50 (0-72 h): 0.49 mg/L
EyC50 (0-72 h): 0.18 mg/L, 95 % confidence limits 0.16 - 0.21 mg/L
NOEC (gowth rate): 0.0625 mg/L
NOEC (yield) 0.0625 mg/L
LOEC (growth rate): 0.125 mg/L
LOEC (Yield): 0.125 mg/L
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concrntrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Growth Rate
Statistical analysis of the growth rate was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant decreases in growth rate between the control, 10, 20, 40 and 80 mg/L test concentrations (P >=0.05), however the 160 mg/L test concentration was significantly different (P<0.05) and , therefore the "No Obseerved Effect Concentration" (NOEC) based on growth rate was 80 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 160 mg/L.

Inhibition of Yield:
Statistical analysis of the yield data was carried out as aboce. There were no statistically significant decreases in yield between the control, 10, 20, 40 and 80 mg/L test concentrations (P>=0.05), however the 160 mg/L test concentration was significantly different (P<0.05) and, therefore the NOEC based on yield was 80 mg/L. Correspondingly the LOEC based on yield was 160 mg/L.

Analytical results

Nominal conc. (mg/L)

Measured concentration (mg/L)

Geometric mean measured (mg/L)

% of nominal

0 hours

72 hours

Control

<LOQ

<LOQ

-

-

10

9.17

0.113

1.0

10

20

19.3

2.59

7.1

36

40

38.7

14.0

23

58

80

76.5

35.9

52

65

160

159

80.4

113

71

Based on the analyitcal results it is considered appropriate to based the effects of the study on the geometric mean measured concentrations.

 Algal cell densities

Mean measured conc. (mg/L)

Mean algal cell density (x10000 cell/mL)

24 hours

48 hours

72 hours

Control

11.1

23.6

102

1.0

10.7

24.7

97.9

7.1

9.26

21.9

114

23

6.70

20.9

103

52

7.58

23.0

137

113

4.68

10.3

24.2

 

Inhibition of growth rate and yield

Mean measured conc. (mg/L)

Growth rate (cells/mL/hour)
(0 – 72 hours)

% inhibition

Yield (cells/mL)
 (0 – 72 hours)

% inhibition

Control

0.045

-

9.76E+04

-

1.0

0.045

1

9.37E+04

4

7.1

0.047

[3]

1.10E+05

[12]

23

0.045

[1]

9.86E+04

[1]

52

0.049

[9]

1.33E+05

[36]

113

0.025

44

1.09E+04

80

 

Validity criteria fulfilled:
yes
Conclusions:
The effect of benzyl acetate on the growth of Desmodesmus subspicatus has been investigated over a 72-hour period and gave the following results:
Exposure of Desmodesmus subspicatus to the test item gave the following results:
Growth Rate: EC50 160 mg/L; NOEC: 80 mg/L; LOEC: 160 mg/L
Yield: EC50: 130 mg/L; NOEC: 80 mg/L; LOEC 160 mg/L

Based on the geometric mean measured test concetrations exposure of Desmodesmus subspicatus to the test item gave the following results:
Growth rate: 72 h EC50: 110 mg/L; NOEC: 52 mg/L; LOEC: 113 mg/L
Yield: 72 h EC50: 92 mg/L; NOEC: 52 mg/L; LOEC: 113 mg/L
Executive summary:

A study was performed to assess the effects of benzyl acetate growth of the alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 201.

Nominal exposure concentrations were 10, 20, 40, 80 and 160 mg/L.  Initial measured exposure concentrations were between 92 and 100% of nominal, measured concentrations after 72 hours had declined to between 1 and 50% of nominal. The geometric means were calculated to be 1.0, 7.1, 23, 52 and 113 mg/L. The results of the study are based on the geometric mean exposure concentrations. 

Growth rate: 72 h EC50: 110 mg/L; NOEC: 52 mg/L; LOEC: 113 mg/L

Yield: 72 h EC50: 92 mg/L; NOEC: 52 mg/L; LOEC: 113 mg/L

Description of key information

A study was performed to assess the effects of benzyl acetate growth of the alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 201. 
Nominal exposure concentrations were 10, 20, 40, 80 and 160 mg/L. Initial measured exposure concentrations were between 92 and 100% of nominal, measured concentrations after 72 hours had declined to between 1 and 50% of nominal. The geometric means were calculated to be 1.0, 7.1, 23, 52 and 113 mg/L. The results of the study are based on the geometric mean exposure concentrations.
Growth rate: 72 h EC50: 110 mg/L; NOEC: 52 mg/L; LOEC: 113 mg/L
Yield: 72 h EC50: 92 mg/L; NOEC: 52 mg/L; LOEC: 113 mg/L

Key value for chemical safety assessment

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

Additional information

The effects of benzyl acetate on the growth of the alga Desmodesmus subspicatus were investigated (Vyrenhoef, H. and Mullee, D.M. 2010). Growth rate: 72 h EC50: 110 mg/L; NOEC: 52 mg/L; LOEC: 113 mg/L Yield: 72 h EC50: 92 mg/L; NOEC: 52 mg/L; LOEC: 113 mg/L.